Immunogenic compositions

ABSTRACT

The present invention relates fusion partners which act as immunological fusion partners, as expression enhancers, and preferably to fusion partners having both functions. In particular the fusion partners contain a so-called choline binding domain, for example fusions comprising LytA from  Streptococcus pneumoniae , or the pneumococcal phage CP1 lysozyme (CPL1) wherein the choline binding domain is modified to include a heterologous T-helper epitope, and are fused to antigens, particularly poorly immunogenic antigens such as self-antigens, eg tumour specific or tissue specific antigens. The invention also relates to fusion proteins containing them, to their manufacture, to their use in immunogenic compositions and vaccines and to their use in medicines.

The present invention relates to fusion partners which act asimmunological fusion partners, as expression enhancers, and preferablyto fusion partners having both functions. The invention also relates tofusion proteins containing them, to their manufacture, to their use invaccines and to their use in medicines. In particular fusion partnersare provided that contain a so-called choline binding domain, forexample fusions comprising LytA from Streptococcus pneumoniae, or thepneumococcal phage CP1 lysozyme (CPL1) wherein the choline bindingdomain is modified to include a heterologous T-helper epitope. Suchfusion partners are shown to improve the expression level of theheterologous protein attached thereto and also find particular utilitywhen fused to poorly immunogenic proteins or peptides that are otherwiseuseful as vaccine antigens. More particularly, such fusion partners areuseful in constructs comprising self-antigens, eg tumour specific ortissue specific antigens.

BACKGROUND TO THE INVENTION

Streptococcus pneumoniae synthesises an N acetyl-L-alanine amidase,LytA, an autolysin that specifically degrades the peptidoglycan backboneof the cell wall eventually leading to cell lysis. Its polypeptide chainhas two domains. The N-terminal domain is responsible for the catalyticactivity, whereas the C-terminal domain of LytA is responsible for theaffinity to choline and anchorage to the cell wall. This C-terminaldomain is known to bind to choline and choline analogues, and will alsobind to tertiary amines such as DEAE (diethyl amino ethyl) commonly usedin chromatography.

LytA is a 318 amino acid protein, and the C-terminal part comprises atandem of six imperfect repeats of 20 or 21 amino acids and a shortCOOH-terminal tail. The repeats are located at the following positions:

R1: 177-191

R2: 192-212

R3: 213-234

R4: 235-254

R5: 255-275

R6: 276-298

These repeats are predicted to be in a beta-turn conformation. TheC-terminus is responsible for binding choline. Likewise the C-terminusof CPL1 is responsible for binding affinity and the aromatic residues inthe repeat contribute to such binding. These proteins have been used asaffinity tags to allow for rapid purification (Sanchez Puelles, Eur JBiochem. 1992, 203, 153-9).

Other proteins with a choline-binding domain have also been studied inStreptococcus pneumoniae.

One of them PspA (or Pneumococcal Surface Protein A), is a virulencefactor (Yother J and Briles (1992) J Bacteriol 174(2) p 601). Thisprotein is antigenic and immunogenic. It has a C-terminal domainconsisting of 10 repeats of 20 amino acids, homologous with repeats ofLytA.

CbpA (or Choline-Binding Protein A) is involved in the adherence of thepneumococcus to human cells (Rosenow et al (1997) Mol Microbiol 25 (5) p819). It shows 10 repeats of 20 amino acids in the C-terminal domainwhich are almost identical to those of PspA.

LytB and LytC have a different modular organisation from theabove-mentioned proteins as their choline-binding domain, made up of 15repeats and 11 repeats respectively, is situated at the N-terminal end,not at the C-terminal end (Garcia P Mol Microbiol (1999) 31 (4) p1275and Garcia P et al (1999) Mol Microbiol 33(1) p128). Sequence comparisonshows LytB to have glucosamidase activity. LytC shows in vitro alysozyme-type activity. Additionally, three genes called PepA, PepB andPepC were cloned in 1995. Although their function is unknown, thesegenes also have a variable number of repeats homologous to those ofLytA.

In their infection cycle, phages synthesise murein hydrolasesfacilitating their passage into the bacterium. These hydrolases have acholine-binding domain.

The muramidase CPL1 of the phage Cp-1 has been well studied. It shows 6repeats of 20 amino acids at the C-terminus involved in the specificrecognition of choline (Garica J. L. J. Virol 61 (8) p2573-80; (1987)and Garcia E Prol Natl Acad Sci (1988) p914). A comparison of the LytAand CPL1 repeats enables an initial consensus of those repeats to bemade.

The murein hydrolases of phages Dp-1 (Garcia P et al (1983) J GenMicrobiol 129 (2) p489, Cpl-9 (Garcia P et al (1989) Biochem Biophys ResCommun 158(1) p 251, HB-3 Romero et al 1990 J Bacteriol 172 (9) p5064-5070) and EJ-1 Diaz (1992) J Bacteriol 174 (17) p 5516), also showthe characteristics of choline-binding domains.

This property is also shared by the lysozyme encoded by CP-1 apneumococal phage. WO 99/10375 describes inter alia, human papillomavirus proteins E6, or E7 linked to a His tag and the C-terminal portionof LytA (herein (C-LytA) and the purification of the proteins bydifferential affinity chromatography.

WO 99/40188 describes inter alia fusion proteins comprising MAGEantigens with a His tails and a C-LytA portion at the N-terminus of themolecule.

It has now been surprisingly found that fusion partners according to thepresent invention, when fused to a heterologous protein were capable ofenhancing the immunogenicity of the heterologous proteins attachedthereto. It has also been found that the expression level of theheterologous proteins attached thereto can be enhanced. The presentinvention accordingly provides in a preferred embodiment an improvedimmunological fusion partner which can also act as an expressionenhancer.

SUMMARY OF THE INVENTION

Accordingly the present invention comprises a fusion partner moleculecomprising a choline binding domain or a fragment thereof or an analoguethereof, and a heterologous promiscuous T helper epitope, preferably apromiscuous MHC Class II T-epitope. Said fusion partner shows acapability of acting as both an immunological fusion partner, or as anexpression enhancer and preferably as both an immunological partner andexpression enhancer. A promiscuous T-helper epitope is an epitope thatbinds to more than one MHC Class II allele, preferably more than 3 MHCClass II alleles. In particular such epitopes are capable of elicitinghelper T cell response in large numbers of individuals expressingdiverse MHC haplotypes. Optionally, the fusion protein may retain itscapability to bind to choline.

In a preferred embodiment the choline binding moiety is derived from theC terminus of LytA. Preferably the C-LytA or derivatives comprises atleast four repeats of any of the repeats R1 to R6 set forth in FIG. 1(SEQ ID NO:1 to 6). In a most preferred embodiment, the C-LytA extendsfrom amino acid 177-298 which contains a portion of the first repeat andthe complete five others.

In a further aspect of the invention, there is provided a fusion partneras herein defined further comprising a heterologous protein. Theheterologous protein may be either chemically conjugated or fused to thefusion partner. Preferably the heterologous protein is atumour-associated antigen or immunogenic fragment thereof.

In a further aspect of the invention there is provided a nucleic acidsequence encoding the proteins as herein defined. There is also providedan expression vector comprising said nucleic acid, and a hosttransformed with said nucleic acid or vector.

In a further aspect of the invention there is provided an immunogeniccomposition comprising a protein or a nucleic acid sequence as hereindescribed, and a pharmaceutically acceptable excipient, diluent orcarrier. Preferably the immunogenic composition further comprises a Th-1inducing adjuvant.

In yet a further embodiment, the invention provides the immunogeniccomposition or protein and nucleic acids for use in medicine. Inparticular, there is provided a protein or a nucleic acid of theinvention, in the manufacture of a medicament for eliciting an immuneresponse in a patient, or for use in the treatment or prophylaxis ofinfectious diseases or cancer diseases.

The invention further provides for methods of treating a patientsuffering from an infectious disease or a cancer disease, particularlycarcinoma of the breast, lung (particularly non-small cell lungcarcinoma), colorectal, ovarian, prostate, gastric and other GI(gastrointestinal) by the administration of a safe and effective amountof a composition or nucleic acid as herein described.

In yet a further embodiment the invention provides a method of producingan immunogenic composition as herein described by admixing a nucleicacid or protein of the invention with a pharmaceutically acceptableexcipient, diluent or carrier.

DETAILED DESCRIPTION OF THE INVENTION

As described therein, in one embodiment of the present invention themodified choline binding domain (fusion partner) has a capability ofacting as an expression enhancer with the resulting fusion protein willbe expressed at a higher yield in a host cell as compared to the unfusedprotein, preferably at a yield greater than about 100% (2-fold higher)or 150% or more, as measured by SDS-PAGE followed by Coomassie bluestaining or silver staining, optionally followed by gel scanning. Themodified choline binding domain according to the invention has also thecapability of acting as an immunological partner with the resultingfusion protein with a heterologous protein will be more immunogenic in ahost as compared to the unfused heterologous protein.

In another embodiment of the present invention, the modified cholinebinding domain has the capability to act as an immunological fusionpartner, allowing an enhanced immune response to be obtained with thefusion protein as compared to the heterologous protein alone.

In a preferred embodiment, the modified choline binding domain has adual function, having the capability to act as both an immunologicalfusion partner and as an expression enhancer.

In a preferred embodiment the choline binding moiety is derived from theC terminus of LytA. Preferably the C-LytA or derivatives comprises atleast two repeats, preferably at least four repeats. In this context,C-LytA derivatives refer to a variant of C-LytA according to the presentinvention, that is to say variants which have retained both thecapability of acting as an immunological partner and an expressionenhancer. Preferred variants include, for example, peptides comprisingan amino acid sequence having at least 85% identity, preferably at least90% identity, more preferably at least 95% identity, most preferably atleast 97-99% identity, to any of the repeats R1 to R6 set forth in FIG.1 (SEQ ID NO:1 to 6), or a peptide comprising an amino acid sequencehaving at least 15, 20, 30, 40, 50 or 100 contiguous amino acids fromthe amino acid sequence set forth in FIG. 1 (SEQ ID NO:1 to 8).

Accordingly, in one aspect of the invention there is provided a fusionpartner protein comprising a modified choline binding domain and aheterologous promiscuous T helper epitope, wherein the choline bindingdomain is selected from the group comprising:

-   -   a) the C-terminal domain of LytA as set forth in SEQ ID NO:7;    -   b) the sequence of SEQ ID NO:8;    -   c) a peptide sequence comprising an amino acid sequence having        at least 85% identity, preferably at least 90% identity, more        preferably at least 95% identity, most preferably at least        97-99% identity, to any of SEQ ID NO:1 to 6;    -   d) a peptide sequence comprising an amino acid sequence having        at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from        the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

In a most preferred embodiment, the C-LytA extends from amino acid177-298 which contains a portion of the first repeat and the completefive others, as set forth in FIG. 1.

The second component of the fusion partner, the heterologous T-cellepitope is preferably selected from the group of epitopes that will bindto a number of individuals expressing more than one MHC II molecules inhumans. For example, epitopes that are specifically contemplated are P2and P30 epitopes from tetanus toxoid, Panina-Bordignon Eur. J. Immunol19 (12), 2237 (1989). In a preferred embodiment the heterologous T-cellepitope is P2 or P30 from Tetanus toxin.

The P2 epitope has the sequence QYIKANSKFIGITE and corresponds to aminoacids 830-843 of the Tetanus toxin. The P30 epitope (residues 947-967 ofTetanus Toxin) has the sequence FNNFTVSFWLRVPKVSASHLE. The FNNFTVsequence may optionally be deleted. Other universal T epitopes can bederived from the circumsporozoite protein from Plasmodium falciparum—inparticular the region 378-398 having the sequence DIEKKIAKMEKASSVFNWNS(Alexander J, (1994) Immunity 1 (9), p 751-761). Another epitope isderived from Measles virus fusion protein at residue 288-302 having thesequence LSEIKGVIVHRLEGV (Partidos C D, 1990, J. Gen. Virol 71(9)2099-2105). Yet another epitope is derived from hepatitis B virussurface antigen, in particular amino acids, having the sequenceFFLLTRILTIPQSLD. Another set of epitopes is derived from diphteriatoxin. Four of these peptides (amino acids 271-290, 321-340, 331-350,351-370) map within the T domain of fragment B of the toxin, and theremaining 2 map in the R domain (411-430, 431-450):

PVFAGANYAAWAVNVAQVI

VHHNTEEIVAQSIALSSLMV

QSIALSSLMVAQAIPLVGEL

VDIGFMYNFVESII NLFQV

QGESGHDIKITAENTPLPIA

GVLLPTIPGKLDVNKSKTHI

(Raju R., Navaneetham D., Okita D., Diethelm-Okita B., McCormick D.,Conti-Fine B. M. (1995) Eur. J. Immunol. 25: 3207-14.)

The heterologous T-epitope is preferably fused to C-LytA containing atleast 4 repeats, preferably repeat 2-5 inclusive. One or more subsequentrepeats may optionally be fused to the C-terminus of the T-epitope.Alternatively, the heterologous T-epitope is preferably inserted betweentwo consecutive repeats of C-LytA containing a total of at least 4repeats, or inserted into one of the repeats of C-LytA containing atotal of at least 4 repeats. More preferably, the C-LytA contains 6repeats and the heterologous epitope is inserted within and at thebeginning of the sixth repeat of C-LytA.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant.

Thus a self-protein or other poorly immunogenic protein may be fused toeither the N or C terminal end of the resulting fusion partner.Alternatively the self protein or poorly immunogenic protein may beinserted into the fusion partner. In an optional embodiment a histidinetag or at least four, preferably more than 6 histidine residues, may befused to the alternative end of the poorly immunogenic protein. Thiswould allow for the protein to be purified by affinity chromatographysteps, as a histidine tail, typically comprising at least four,preferably six or more residues binds to metal ions and therefore issuitable for metal immobilised metal ion affinity chromatography (IMAC).

Typical constructs would therefore comprise:

-   -   Poorly-immunogenic protein—C-LytA repeats₁₋₄-P₂ epitope        (inserted in or replacing C-LytA repeat₅)-C-LytA repeat₆    -   C-LytA repeats₁₋₄-P₂ epitope (inserted in or replacing C-LytA        repeat₅)—C-LytA repeat₆—Poorly immunogenic protein    -   Poorly immunogenic protein—C-LytA repeat₂₋₅-P₂ epitope (inserted        into C-LytA repeat₆₎    -   C-LytA₂₋₅-P₂epitope (inserted into C-LytA repeats)—Poorly        immunogenic protein.    -   Poorly immunogenic protein C-LytA repeats₁₋₅-P₂ epitope-inserted        in C-LytA repeat    -   C-LytA repeats₁₋₅-P₂ epitope-inserted in C-LytA repeat₆—Poorly        immunogenic protein    -   Poorly immunogenic protein-P₂ epitope inserted into C-LytA        repeat₁-C-LytA repeats₂₋₅    -   P₂ epitope inserted into C-LytA repeat₁-C-LytA repeats₂₋₅—Poorly        immunogenic protein    -   Poorly immunogenic protein-P₂ epitope inserted into C-LytA        repeat₁-C-LytA repeats₂₋₆    -   P₂ epitope inserted into C-LytA repeat₁-C-LytA repeats₂₋₈-Poorly        immunogenic protein    -   Poorly immunogenic protein-C-LytA repeat₁-P₂ epitope inserted        into C-LytA repeat₂-C-LytA repeats₃₋₆    -   C-LytA repeat₁-P₂ epitope inserted into C-LytA repeat₂-C-LytA        repeats₃₋₆-Poorly immunogenic protein;        where “inserted into” means at any place into the repeat for        example between residue 1 and 2, or between 2 and 3, etc.

The promiscuous T helper epitope may be inserted within a repeat regionfor example C-LytA repeats₂₋₅ _(—) -C-LytA repeat 6a-P₂ epitope—C-LytArepeat 6b, where the P2 epitope is inserted within the sixth repeat (seeFIG. 2).

In other preferred embodiments the C-terminal end of CPL1 (C-CPL1) maybe used as an alternative to C-LytA.

Alternatively, the P2 epitope in the above constructs may be replaced byother promiscuous T epitopes, for example P30. In an embodiment of theinvention, two or more promiscuous epitopes are part of the fusionconstruct. It is however preferred to keep the fusion partner as smallas possible, thus limiting the number of potentially interfering CD8+and B epitopes. Thus the fusion partner is preferably no bigger than100-140 amino acids, preferably no bigger than 120 amino acids,typically about 100 amino acid.

The antigen to which the fusion partner is fused may be from bacterial,viral, protozoan, fungal or mammalian, including human, sources.

The fusion partner of the present invention are preferably fused to aself antigen such as a tumour associated or tissue specific antigenssuch as those for prostrate, breast, colorectal, lung, pancreatic,ovarian, renal or melanoma cancers. Fragments of said self or tumourantigens are expressly contemplated to be fused to the fusion partner ofthe invention. Typically the fragment will contain at least 20,preferably 50, more preferably 100 contiguous amino acids of thefull-length sequence. Typically such fragments will be devoid of one ormore transmembrane domains or may have N-terminal or C-terminaldeletions of about 3, 5, 8, 10, 15, 20, 28, 33, 50, 54 amino acids. Suchfragments will, when suitably presented, be able to generate immuneresponses that recognise the full length protein. Particularlyillustrative polypeptides of the present invention comprise a sequenceof at least 10 contiguous amino acids, preferably 20, more preferably30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180amino acids of a tumour associated or tissue specific protein fused tothe fusion partner.

The polypeptides of the invention are immunogenic, i.e., they reactdetectably within an immunoassay (such as an ELISA or T-cell stimulationassay) with antisera and/or T-cells from a patient with criptoexpressing cancer. Screening for immunogenic activity can be performedusing techniques well known to the skilled artisan. For example, suchscreens can be performed using methods such as those described in Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In one illustrative example, a polypeptide may beimmobilised on a solid support and contacted with patient sera to allowbinding of antibodies within the sera to the immobilised polypeptide.Unbound sera may then be removed and bound antibodies detected using,for example, ¹²⁵I-labeled Protein A. As would be recognised by theskilled artisan, immunogenic portions of tumour associated or tumourspecific antigen are also encompassed by the present invention. An“immunogenic portion” as used herein, is a fragment that itself isimmunologically reactive (i.e., specifically binds) with the B-cellsand/or T-cell surface antigen receptors that recognize the polypeptide.Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques. In one preferred embodiment, animmunogenic portion of a polypeptide is a portion that reacts withantisera and/or T-cells at a level that is not substantially less thanthe reactivity of the full-length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Preferably, the level of immunogenic activityof the immunogenic portion is at least about 50%, preferably at leastabout 70% and most preferably greater than about 90% of theimmunogenicity for the full-length polypeptide. In some instances,preferred immunogenic portions will be identified that have a level ofimmunogenic activity greater than that of the corresponding full-lengthpolypeptide, e.g., having greater than about 100% or 150% or moreimmunogenic activity.

In certain other embodiments, illustrative immunogenic portions mayinclude peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., about1-50 amino acids, preferably about 1-30 amino acids, more preferablyabout 5-15 amino acids), relative to the mature protein.

Exemplary antigens or fragments derived therefrom include MAGE 1, Mage 3and MAGE 4 or other MAGE antigens such as disclosed in WO 99/40188,PRAME (WO 96/10577), BAGE, RAGE, LAGE 1 (WO 98/32855), LAGE 2 (alsoknown as NY-ESO-1, WO 98/14464), XAGE (Liu et al, Cancer Res, 2000,60:4752-4755; WO 02/18584) SAGE, and HAGE (WO 99/53061) or GAGE (Robbinsand Kawakami, 1996, Current Opinions in Immunology 8, pps 628-636; Vanden Eynde et al., International Journal of Clinical & LaboratoryResearch (submitted 1997); Correale et al. (1997), Journal of theNational Cancer Institute 89, p293. Indeed these antigens are expressedin a wide range of tumour types such as melanoma, lung carcinoma,sarcoma and bladder carcinoma.

In a preferred embodiment prostate antigens are utilised, such asProstate specific antigen (PSA), PAP, PSCA (PNAS 95(4) 1735-1740 1998),PSMA or the antigen known as prostase.

In a particularly preferred embodiment, the prostate antigen is P501S ora fragment thereof. P501S, also named prostein (Xu et al., Cancer Res.61, 2001, 1563-1568), is known as SEQ ID NO. 113 of WO98/37814 and is a553 amino acid protein. Immunogenic fragments and portions thereofcomprising at least 20, preferably 50, more preferably 100 contiguousamino acids as disclosed in the above referenced patent application andare specifically contemplate by the present invention. Preferredfragments are disclosed in WO 98/50567 (PS108 antigen) and as prostatecancer-associated protein (SEQ ID NO: 9 of WO 99/67384). Other preferredfragments are amino acids 51-553, 34-553 or 55-553 of the full-lengthP501S protein. In particular, construct 1, 2 and 3 (see FIG. 2, SEQ IDNOs. 27-32) are expressly contemplated, and can be expressed in yeastsystems, for example DNA sequences encoding such polypeptides can beexpressed in yeast system.

Prostase is a prostate-specific serine protease (trypsin-like), 254amino acid-long, with a conserved serine protease catalytic triad H-D-Sand a amino-terminal pre-propeptide sequence, indicating a potentialsecretory function (P. Nelson, Lu Gan, C. Ferguson, P. Moss, R. Iinas,L. Hood & K. Wand, “Molecular cloning and characterisation of prostase,an androgen-regulated serine protease with prostate restrictedexpression, In Proc. Natl. Acad. Sci. USA (1999) 96, 3114-3119). Aputative glycosylation site has been described. The predicted structureis very similar to other known serine proteases, showing that the maturepolypeptide folds into a single domain. The mature protein is 224 aminoacids-long, with one A2 epitope shown to be naturally processed.Prostase nucleotide sequence and deduced polypeptide sequence andhomologous are disclosed in Ferguson, et al. (Proc. Natl. Acad. Sci. USA1999, 96, 3114-3119) and in International Patent Applications No. WO98/12302 (and also the corresponding granted patent U.S. Pat. No.5,955,306), WO 98/20117 (and also the corresponding granted patents U.S.Pat. No. 5,840,871 and U.S. Pat. No. 5,786,148) (prostate-specifickallikrein) and WO 00/04149 (P703P).

Other prostate specific antigens are known from WO98/37418, andWO/004149. Another is STEAP (PNAS 96 14523 14528 7-12 1999).

Other tumour associated antigens useful in the context of the presentinvention include: Plu −1 J. Biol. Chem 274 (22) 15633-15645, 1999,HASH-1, HASH-2 (Alders, M. et al., Hum. Mol. Genet. 1997, 6, 859-867),Cripto (Salomon et al Bioessays 199, 21 61-70, U.S. Pat. No. 5,654,140),CASB616 (WO 00/53216), Criptin (U.S. Pat. No. 5,981,215). Additionally,antigens particularly relevant for vaccines in the therapy of canceralso comprise tyrosinase, telomerase, P53, NY-Br1.1 (WO 01/47959) andfragments thereof such as disclosed in WO 00/43420, B726 (WO 00/60076,SEQ ID nos 469 and 463; WO 01/79286, SEQ ID nos 474 and 475), P510 (WO01/34802 SEQ ID nos 537 and 538) and survivin.

The present invention is also useful in combination with breast cancerantigens such as Her-2/neu, mammaglobin (U.S. Pat. No. 5,668,267), B305D(WO 00/61753 SEQ ID nos 299, 304, 305 and 315), or those disclosed in WO00/52165, WO 99/33869, WO 99/19479, WO 98/45328. Her-2/neu antigens aredisclosed inter alia, in U.S. Pat. No. 5,801,005. Preferably theHer-2/neu comprises the entire extracellular domain (comprisingapproximately amino acid 1-645) or fragments thereof and at least animmunogenic portion of or the entire intracellular domain approximatelythe C terminal 580 amino acids. In particular, the intracellular portionshould comprise the phosphorylation domain or fragments thereof. Suchconstructs are disclosed in WO 00/44899. A particularly preferredconstruct is known as ECD-PhD, a second is known as ECD deltaPhD (see WO00/44899). The Her-2/neu as used herein can be derived from rat, mouseor human.

Certain tumour antigens are small peptide antigens (ie less than about50 amino acids). These antigens can be chemically conjugated to themodified choline binding protein of the present invention.

Exemplary peptides included Mucin derived peptides such as MUC-1 (seefor example U.S. Pat. No. 5,744,144; U.S. Pat. No. 5,827,666; WO88/05054, U.S. Pat. No. 4,963,484). Specifically contemplated are MUC-1derived peptides that comprise at least one repeat unit of the MUC-1peptide, preferably at least two such repeats and which is recognised bythe SM3 antibody (U.S. Pat. No. 6,054,438). Other mucin derived peptidesinclude peptide from MUC-5.

Alternatively, said antigen is an interleukin such as IL13 and IL14,which are preferred. Or said antigen maybe a self peptide hormone suchas whole length Gonadotrophin hormone releasing hormone (GnRH, WO95/20600), a short 10 amino acid long peptide, useful in the treatmentof many cancers, or in immunocastration.

Other tumour-specific antigens are suitable to be coupled with themodified Choline binding protein of the present invention include, butare not restricted to tumour-specific gangliosides such as GM2, and GM3.

The covalent coupling of the peptide to modified choline binding proteincan be carried out in a manner well known in the art. Thus, for example,for direct covalent coupling it is possible to utilise a carbodiimide,glutaraldehyde or (N-[γ-maleimidobutyryloxy]succinimide ester, utilisingcommon commercially available heterobifunctional linkers such as CDAPand SPDP (using manufacturers instructions). After the couplingreaction, the immunogen can easily be isolated and purified by means ofa dialysis method, a gel filtration method, a fractionation method etc.

The antigen may also be derived from sources which are pathogenic tohumans, such as such as Human Immunodeficiency virus HIV-1 (such as tat,nef, reverse transcriptase, gag, gp120 and gp160), human herpes simplexviruses, such as gD or derivatives thereof or Immediate Early proteinsuch as ICP27 from HSV1 or HSV2, cytomegalovirus ((esp Human)(such as gBor derivatives thereof, Rotavirus (including live-attenuated viruses),Epstein Barr virus (such as gp350 or derivatives thereof), VaricellaZoster Virus (such as gpl, II and IE63), or from a hepatitis virus suchas hepatitis B virus (for example Hepatitis B Surface antigen or aderivative thereof), hepatitis A virus, hepatitis C virus and hepatitisE virus, or from other viral pathogens, such as paramyxoviruses:Respiratory Syncytial virus (such as F and G proteins or derivativesthereof), parainfluenza virus, measles virus, mumps virus, humanpapilloma viruses (for example HPV6, 11, 16, 18, . . . ), flaviviruses(e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,Japanese Encephalitis Virus) or Influenza virus (whole live orinactivated virus, split influenza virus, grown in eggs or MDCK cells,or whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10,915-920) or purified or recombinant proteins thereof, such as HA, NP,NA, or M proteins, or combinations thereof), or derived from bacterialpathogens such as Neisseria spp, including N. gonorrhea and N.meningitidis (for example capsular polysaccharides and conjugatesthereof, transferrin-binding proteins, lactoferrin binding proteins,PilC, adhesins); S. pyogenes (for example M proteins or fragmentsthereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans; H.ducreyi; Moraxella spp, including M catarrhalis, also known asBranhamella catarrhalis (for example high and low molecular weightadhesins and invasins); Bordetella spp, including B. pertussis (forexample pertactin, pertussis toxin or derivatives thereof, filamenteoushemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B.bronchiseptica; Mycobacterium spp., including M. tuberculosis (forexample ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M.paratuberculosis, M. smegmatis; Legionella spp, including L.pneumophila; Escherichia spp, including enterotoxic E. coli (for examplecolonization factors, heat-labile toxin or derivatives thereof,heat-stable toxin or derivatives thereof), enterohemorragic E. coli,enteropathogenic E. coli (for example shiga toxin-like toxin orderivatives thereof); Vibrio spp, including V. cholera (for examplecholera toxin or derivatives thereof); Shigella spp, including S.sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y.enterocolitica (for example a Yop protein), Y. pestis, Y.pseudotuberculosis; Campylobacter spp, including C. jejuni (for exampletoxins, adhesins and invasins) and C. coli; Salmonella spp, including S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Usteria spp.,including L. monocytogenes; Helicobacter spp, including H. pylori (forexample urease, catalase, vacuolating toxin); Pseudomonas spp, includingP. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis;Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,including C. tetani (for example tetanus toxin and derivative thereof),C. botulinum (for example botulinum toxin and derivative thereof), C.difficile (for example clostridium toxins A or B and derivativesthereof); Bacillus spp., including B. anthracis (for example botulinumtoxin and derivatives thereof); Corynebacterium spp., including C.diphtheriae (for example diphtheria toxin and derivatives thereof);Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA,DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (forexample OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC,DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agentof the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; Chlamydia spp., including C. trachomatis (for example MOMP,heparin-binding proteins), C. pneumoniae (for example MOMP,heparin-binding proteins), C. psittaci; Leptospira spp., including L.interrogans; Treponema spp., including T. pallidum (for example the rareouter membrane proteins), T. denticola, T. hyodysenteriae; or derivedfrom parasites such as Plasmodium spp., including P. falciparum;Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34);Entamoeba spp., including E. histolytica; Babesia spp., including B.microti; Trypanosoma spp., including T. cruzi; Giardia spp., includingG. lamblia; Leshmania spp., including L. major; Pneumocystis spp.,including P. carini; Trichomonas spp., including T. vaginalis;Schisostoma spp., including S. mansoni, or derived from yeast such asCandida spp., including C. albicans; Cryptococcus spp., including C.neoformans.

Other preferred specific antigens for M. tuberculosis are for example TbRa12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1 (WO99/51748). Proteins for M. tuberculosis also include fusion proteins andvariants thereof where at least two, preferably three polypeptides of M.tuberculosis are fused into a larger protein. Preferred fusions includeRa12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14-DPV-MTI-MSL-mTCC2,Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9-DPV-MTI (WO 99/51748).

Most preferred antigens for Chlamydia include for example the HighMolecular Weight Protein (HWMP) (WO 99/17741), ORF3 (EP 366 412), andputative membrane proteins (Pmps). Other Chlamydia antigens of thevaccine formulation can be selected from the group described in WO99/28475.

Preferred bacterial antigens are derived from Streptococcus spp,including S. pneumoniae (for example capsular polysaccharides andconjugates thereof, PsaA, PspA, streptolysin, choline-binding proteins)and the protein antigen Pneumolysin (Biochem Biophys Acta, 1989, 67,1007; Rubins et al., Microbial Pathogenesis, 25, 337-342), and mutantdetoxified derivatives thereof (WO 90/06951; WO 99/03884). Otherpreferred bacterial antigens are derived from Haemophilus spp.,including H. influenzae type B (for example PRP and conjugates thereof),non typeable H. influenzae, for example OMP26, high molecular weightadhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrinderived peptides (U.S. Pat. No. 5,843,464) or multiple copy varients orfusion proteins thereof.

Derivatives of Hepatitis B Surface antigen are well known in the art andinclude, inter alia, those PreS1, PreS2 S antigens set forth describedin European Patent applications EP-A-414 374; EP-A-0304 578, and EP198474. In one preferred The HBV antigen is HBV polymerase (Ji HoonJeong et al, 1996, BBRC 223, 264-271; Lee H. J. et al, Biotechnol. Lett.15, 821-826). In another preferred aspect the antigen within the fusionis a HIV-1 antigen, gp120, especially when expressed in CHO cells. In afurther embodiment, antigen comprises gD2t as hereinabove defined.

In a preferred embodiment of the present invention fusions comprise anantigen derived from the Human Papilloma Virus (HPV 6a, 6b, 11, 16, 18,31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68), in particular those HPVserotypes considered to be responsible for genital warts (HPV 6 or HPV11 and others), and the HPV viruses responsible for cervical cancer(HPV16, HPV18 and others).

Suitable HPV antigens are E1, E2, E4, E5, E6, E7, L1 and L2.Particularly preferred forms of genital wart prophylactic, ortherapeutic, fusions comprise L1 particles or capsomers, and fusionproteins comprising one or more antigens selected from the HPV 6 and HPV11 proteins E6, E7, L1, and L2.

The most preferred forms of fusion protein are: L2E7 as disclosed in WO96/26277, and proteinD(1/3)-E7 disclosed in GB 9717953.5(PCT/EP98/05285).

A preferred HPV cervical infection or cancer, prophylaxis or therapeuticvaccine, composition may comprise HPV 16 or 18 antigens. For example, L1or L2 antigen monomers, or L1 or L2 antigens presented together as avirus like particle (VLP) or the L1 alone protein presented alone in aVLP or caposmer structure. Such antigens, virus like particles andcapsomer are per se known. See for example WO94/00152, WO94/20137,WO94/05792, and WO93/02184.

Additional early proteins may be included alone or as fusion proteinssuch as E7, E2 or preferably E5 for example; particularly preferredembodiments of this includes a VLP comprising L1E7 fusion proteins (WO96/11272). Particularly preferred HPV 16 antigens comprise the earlyproteins E6 or E7 in fusion with a protein D carrier to form ProteinD—E6 or E7 fusions from HPV 16, or combinations thereof; or combinationsof E6 or E7 with L2 (WO 96/26277).

Alternatively the HPV 16 or 18 early proteins E6 and E7, may bepresented in a single molecule, preferably a Protein D—E6/E7 fusion.Other fusions optionally contain either or both E6 and E7 proteins fromHPV 18, preferably in the form of a Protein D—E6 or Protein D—E7 fusionprotein or Protein D E6/E7 fusion protein. Fusions may comprise antigensfrom other HPV strains, preferably from strains HPV 31 or 33.

Fusions according to the present invention comprise antigens derivedfrom parasites that cause Malaria. For example, preferred antigens fromPlasmodia falciparum include RTS,S and TRAP. RTS is a hybrid proteincomprising substantially all the C-terminal portion of thecircumsporozoite (CS) protein of P. falciparum linked via four aminoacids of the preS2 portion of Hepatitis B surface antigen to the surface(S) antigen of hepatitis B virus. Its full structure is disclosed in theInternational Patent Application No. PCT/EP92/02591, published underNumber WO 93/10152 claiming priority from UK patent application No.9124390.7. When expressed in yeast RTS is produced as a lipoproteinparticle, and when it is co-expressed with the S antigen from HBV itproduces a mixed particle known as RTS,S. TRAP antigens are described inthe International Patent Application No. PCT/GB89/00895, published underWO 90/01496. A preferred embodiment of the present invention is a fusionwherein the antigenic preparation comprises a combination of the RTS,Sand TRAP antigens. Other plasmodia antigens that are likely candidatesto be components of the fusion are P. faciparum MSP1, AMA1, MSP3, EBA,GLURP, RAP1, RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA,PfEXP1, Pfs25, Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and theiranalogues in Plasmodium spp.

The present invention also provides a polynucleotide encoding the fusionpartner according to the present invention. The invention furtherrelates a polynucleotide that hybridise to the polynucleotide sequenceprovided herein in FIG. 1 (SEQ ID NO:9 to 16). In this regard, theinvention especially relates to polynucleotides that hybridise understringent conditions to the polynucleotide described herein. As hereinused, the terms “stringent conditions” and “stringent hybridisationconditions” mean hybridisation occurring only if there is at least 95%and preferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridisation supportin 0.1×SSC at about 65° C. Hybridisation and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridisation may also be usedwith the polynucleotide sequences provided by the invention.

The present invention also provides a polynucleotide encoding thepolypeptide comprising the fusion partner according to the presentinvention fused to a tumour associated antigen or fragment thereof. Inparticular, the present invention provides for polynucleotide sequencesencoding a fusion partner protein comprising a choline binding domainand a heterologous promiscuous T heper epitope, preferably wherein thecholine binding domain is derived from the C terminus of LytA. In a morepreferred embodiment, the C-LytA moiety of the polynucleotides accordingto the invention comprise at least four repeats of any of SEQ IDNO.9-14, more preferably comprise the sequence of SEQ ID NO.15, stillmore preferably the sequence of SEQ ID NO.16. In other relatedembodiments, the present invention provides for polynucleotide variantshaving substantial identity to the sequences disclosed herein in SEQ IDNOs:9-16, for example those comprising at least 70% sequence identity,preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% orhigher, sequence identity compared to a polynucleotide sequence of thisinvention using conventional methods, e.g., BLAST analysis usingstandard parameters. In a still further embodiment the polynucleotide asclaimed further comprises a heterologous protein.

Such polynucleotide sequences can be inserted into a suitable expressionvector and expressed in a suitable host. Vectors may be provided whichencode the modified choline binding protein of the invention and whichcontain a suitable restriction site into which a DNA encoding a poorlyimmunogenic protein can be inserted to produce a fusion protein. Inother embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptide fusions of the invention, maybe used in recombinant DNA molecules to direct expression of apolypeptide in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other DNA sequences that encode substantially the sameor a functionally equivalent amino acid sequence may be produced andthese sequences may be used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. The DNAcode has 4 letters (A, T, C and G) and uses these to spell three letter“codons” which represent the amino acids the proteins encodes in anorganism's genes. The linear sequence of codons along the DNA moleculeis translated into the linear sequence of amino acids in the protein(s)encoded by those genes. The code is highly degenerate, with 61 codonscoding for the 20 natural amino acids and 3 codons representing “stop”signals. Thus, most amino acids are coded for by more than one codon—infact several are coded for by four or more different codons.

Where more than one codon is available to code for a given amino add, ithas been observed that the codon usage patterns of organisms are highlynon-random. Different species show a different bias in their codonselection and, furthermore, utilisation of codons may be markedlydifferent in a single species between genes which are expressed at highand low levels. This bias is different in viruses, plants, bacteria andmammalian cells, and some species show a stronger bias away from arandom codon selection than others. For example, humans and othermammals are less strongly biased than certain bacteria or viruses. Forthese reasons, there is a significant probability that a mammalian geneexpressed in E. coli or a viral gene expressed in mammalian cells willhave an inappropriate distribution of codons for efficient expression.It is believed that the presence in a heterologous DNA sequence ofclusters of codons which are rarely observed in the host in whichexpression is to occur, is predictive of low heterologous expressionlevels in that host.

In consequence, codons preferred by a particular prokaryotic (forexample E. coli or yeast) or eukaryotic host can be optimised, that isselected to increase the rate of protein expression, to produce arecombinant RNA transcript having desirable properties, such as forexample a half-life which is longer than that of a transcript generatedfrom the naturally occurring sequence, or to optimise the immuneresponse in humans. The process of codon optimisation may include anysequence, generated either manually or by computer software, where someor all of the codons of the native sequence are modified. Severalmethods have been published (Nakamura et. al., Nucleic Acids Research1996, 24:214-215; WO98/34640). One preferred method according to thisinvention is Syngene method, a modification of Calcgene method (R. S.Hale and G Thompson (Protein Expression and Purification Vol. 12 pp.185-188 (1998)).

Accordingly in a preferred embodiment the DNA sequence of the proteinhas a RSCU (Relative synomons Codon useage (also known as Codon IndexCI)) of at least 0.65 and have less than 85% identity to thecorresponding wild type region.

This process of codon optimisation and the resulting constructs areadvantageous as they may have some or all of the following benefits: 1)to improve expression of the gene product by replacing rare orinfrequently used codons with more frequently used codons, 2) to removeor include restriction enzyme sites to facilitate downstream cloning and3) to reduce the potential for homologous recombination between theinsert sequence in the DNA vector and genomic sequences and 4) toimprove the immune response in humans by raising a cellular and/or anantibody response (preferably both responses) against the targetantigen. The sequences of the present invention advantageously havereduced recombination potential, but express to at least the same levelas the wild type sequences. Due to the nature of the algorithms used bythe SynGene programme to generate a codon optimised sequence, it ispossible to generate an extremely large number of different codonoptimised sequences which will perform a similar function. In brief, thecodons are assigned using a statistical method to give synthetic genehaving a codon frequency closer to that found naturally in highlyexpressed E. coli and human genes. In brief, the codons are assignedusing a statistical method to give synthetic gene having a codonfrequency closer to that found naturally in highly expressed human genessuch as β-Actin. Illustrative, although non limiting, examples ofsuitable codon-optimised sequences are given in SEQ ID NOs:19-22 and SEQID NOs:24-26.

In the polynucleotides of the present invention, the codon usage patternis altered from that typical of the target antigen to more closelyrepresent the codon bias of a highly expressed gene in a targetorganism, for example human β-actin. The “codon usage coefficient” is ameasure of how closely the codon pattern of a given polynucleotidesequence resembles that of a target species. Codon frequencies can bederived from literature sources for the highly expressed genes of manyspecies (see e.g. Nakamura et. al. Nucleic Acids Research 1996,24:214-215). The codon frequencies for each of the 61 codons (expressedas the number of occurrences occurrence per 1000 codons of the selectedclass of genes) are normalised for each of the twenty natural aminoacids, so that the value for the most frequently used codon for eachamino acid is set to 1 and the frequencies for the less common codonsare scaled to lie between zero and 1. Thus each of the 61 codons isassigned a value of 1 or lower for the highly expressed genes of thetarget species. In order to calculate a codon usage coefficient for aspecific polynucleotide, relative to the highly expressed genes of thatspecies, the scaled value for each codon of the specific polynucleotideare noted and the geometric mean of all these values is taken (bydividing the sum of the natural logs of these values by the total numberof codons and take the anti-log). The coefficient will have a valuebetween zero and 1 and the higher the coefficient the more codons in thepolynucleotide are frequently used codons. If a polynucleotide sequencehas a codon usage coefficient of 1, all of the codons are “mostfrequent” codons for highly expressed genes of the target species.

According to the present invention, the codon usage pattern of thepolynucleotide will preferably exclude codons representing <10% of thecodons used for a particular amino acid. A relative synonymous codonusage (RSCU) value is the observed number of codons divided by thenumber expected if all codons for that amino acid were used equallyfrequently. A polynucleotide of the present invention will preferablyexclude codons with an RSCU value of less than 0.2 in highly expressedgenes of the target organism. A polynucleotide of the present inventionwill generally have a codon usage coefficient for highly expressed humangenes of greater than 0.6, preferably greater than 0.65, most preferablygreater than 0.7. Codon usage tables for human can also be found inGenbank.

In comparison, a highly expressed beta actin gene has a RSCU of 0.747.

The codon usage table (Table 1) for a homo sapiens is set out below:TABLE 1 Codon usage for human (highly expressed) genes Jan. 24, 1991(human_high.cod) AmAcid Codon Number /1000 Fraction Gly GGG 905.00 18.760.24 Gly GGA 525.00 10.88 0.14 Gly GGT 441.00 9.14 0.12 Gly GGC 1867.0038.70 0.50 Glu GAG 2420.00 50.16 0.75 Glu GAA 792.00 16.42 0.25 Asp GAT592.00 12.27 0.25 Asp GAC 1821.00 37.75 0.75 Val GTG 1866.00 38.68 0.64Val GTA 134.00 2.78 0.05 Val GTT 198.00 4.10 0.07 Val GTC 728.00 15.090.25 Ala GCG 652.00 13.51 0.17 Ala GCA 488.00 10.12 0.13 Ala GCT 654.0013.56 0.17 Ala GCC 2057.00 42.64 0.53 Arg AGG 512.00 10.61 0.18 Arg AGA298.00 6.18 0.10 Ser AGT 354.00 7.34 0.10 Ser AGC 1171.00 24.27 0.34 LysAAG 2117.00 43.88 0.82 Lys AAA 471.00 9.76 0.18 Asn AAT 314.00 6.51 0.22Asn AAC 1120.00 23.22 0.78 Met ATG 1077.00 22.32 1.00 Ile ATA 88.00 1.820.05 Ile ATT 315.00 6.53 0.18 Ile ATC 1369.00 28.38 0.77 Thr ACG 405.008.40 0.15 Thr ACA 373.00 7.73 0.14 Thr ACT 358.00 7.42 0.14 Thr ACC1502.00 31.13 0.57 Trp TGG 652.00 13.51 1.00 End TGA 109.00 2.26 0.55Cys TGT 325.00 6.74 0.32 Cys TGC 706.00 14.63 0.68 End TAG 42.00 0.870.21 End TAA 46.00 0.95 0.23 Tyr TAT 360.00 7.46 0.26 Tyr TAC 1042.0021.60 0.74 Leu TTG 313.00 6.49 0.06 Leu TTA 76.00 1.58 0.02 Phe TTT336.00 6.96 0.20 Phe TTC 1377.00 28.54 0.80 Ser TCG 325.00 6.74 0.09 SerTCA 165.00 3.42 0.05 Ser TCT 450.00 9.33 0.13 Ser TCC 958.00 19.86 0.28Arg CGG 611.00 12.67 0.21 Arg CGA 183.00 3.79 0.06 Arg CGT 210.00 4.350.07 Arg CGC 1086.00 22.51 0.37 Gln CAG 2020.00 41.87 0.88 Gln CAA283.00 5.87 0.12 His CAT 234.00 4.85 0.21 His CAC 870.00 18.03 0.79 LeuCTG 2884.00 59.78 0.58 Leu CTA 166.00 3.44 0.03 Leu CTT 238.00 4.93 0.05Leu CTC 1276.00 26.45 0.26 Pro CCG 482.00 9.99 0.17 Pro CCA 456.00 9.450.16 Pro CCT 568.00 11.77 0.19 Pro CCC 1410.00 29.23 0.48

A DNA sequence encoding the fusion proteins or modified choline bindingprotein of the present invention can be synthesised using standard DNAsynthesis techniques, such as by enzymatic ligation as described by D.M. Roberts et al. in Biochemistry 1985, 24, 5090-5098, by chemicalsynthesis, by in vitro enzymatic polymerisation, or by PCR technologyutilising for example a heat stable polymerase, or by a combination ofthese techniques.

Enzymatic polymerisation of DNA may be carried out in vitro using a DNApolymerase such as DNA polymerase I (Klenow fragment) or Taq polymerasein an appropriate buffer containing the nucleoside triphosphates dATP,dCTP, dGTP and dTTP as required at a temperature of 10°-37° C.,generally in a volume of 50 μl or less. Enzymatic ligation of DNAfragments may be carried out using a DNA ligase such as T4 DNA ligase inan appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl₂, 0.01Mdithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg/ml bovine serumalbumin, at a temperature of 4° C. to ambient, generally in a volume of50 μl or less. The chemical synthesis of the DNA polymer or fragmentsmay be carried out by conventional phosphotriester, phosphate orphosphoramidite chemistry, using solid phase techniques such as thosedescribed in ‘Chemical and Enzymatic Synthesis of Gene Fragments—ALaboratory Manual’ (ed. H. G. Gassen and A. Lang), Verlag Chemie,Weinheim (1982), or in other scientific publications, for example M. J.Gait, H. W. D. Matthes, M. Singh, B. S. Sproat, and R. C. Titmas,Nucleic Acids Research, 1982, 10, 6243; B. S. Sproat, and W. Bannwarth,Tetrahedron Letters, 1983, 24, 5771; M. D. Matteucci and M. H.Caruthers, Tetrahedron Letters, 1980, 21, 719; M. D. Matteucci and M. H.Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S.P. Adams et al., Journal of the American Chemical Society, 1983, 105,661; N. D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic AcidsResearch, 1984, 12, 4539; and H. W. D. Matthes et al., EMBO Journal,1984, 3, 801.

The process of the invention may be performed by conventionalrecombinant techniques such as described in Maniatis et al., MolecularCloning—A Laboratory Manual; Cold Spring Harbor, 1982-1989.

In particular, the process may comprise the steps of:

i) preparing a replicable or integrating expression vector capable, in ahost cell, of expressing a DNA polymer comprising a nucleotide sequencethat encodes the protein or an immunogenic derivative thereof

ii) transforming a host cell with said vector

iii) culturing said transformed host cell under conditions permittingexpression of said DNA polymer to produce said protein; and

iv) recovering said protein

The term ‘transforming’ is used herein to mean the introduction offoreign DNA into a host cell. This can be achieved for example bytransformation, transfection or infection with an appropriate plasmid orviral vector using e.g. conventional techniques as described in GeneticEngineering; Eds. S. M. Kingsman and A. J. Kingsman; BlackwellScientific Publications; Oxford, England, 1988. The term ‘transformed’or ‘transformant’ will hereafter apply to the resulting host cellcontaining and expressing the foreign gene of interest.

The expression vectors are novel and also form part of the invention.

The replicable expression vectors may be prepared in accordance with theinvention, by cleaving a vector compatible with the host cell to providea linear DNA segment having an intact replicon, and combining saidlinear segment with one or more DNA molecules which, together with saidlinear segment encode the desired product, such as the DNA polymerencoding the protein of the invention, or derivative thereof, underligating conditions.

Thus, the DNA polymer may be performed or formed during the constructionof the vector, as desired.

The choice of vector will be determined in part by the host cell, whichmay be prokaryotic or eukaryotic but are preferably E. coli, yeast orCHO cells. Suitable vectors include plasmids, bacteriophages, cosmidsand recombinant viruses. Expression and cloning vectors preferablycontain a selectable marker such that only the host cells expressing themarker will survive under selective conditions. Selection genes includebut are not limited to the one encoding protein that confer a resistanceto ampicillin, tetracyclin or kanamycin. Expression vectors also containcontrol sequences which are compatible with the designated host. Forexample, expression control sequences for E. coli, and more generallyfor prokaryotes, include promoters and ribosome binding sites. Promotersequences may be naturally occurring, such as the β-lactamase(penicillinase) (Weissman 1981, In Interferon 3 (ed. L. Gresser),lactose (lac) (Chang et al. Nature, 1977, 198: 1056) and tryptophan(trp) (Goeddel et al. Nucl. Acids Res. 1980, 8, 4057) and lambda-derivedP_(L) promoter system. In addition, synthetic promoters which do notoccur in nature also function as bacterial promoters. This is the casefor example for the tac synthetic hybrid promoter which is derived fromsequences of the trp and lac promoters (De Boer et al., Proc. Natl.Acad. Sci. USA 1983, 80, 21-26). These systems are particularly suitablewith E. coli.

Yeast compatible vectors also carry markers that allow the selection ofsuccessful transformants by conferring prototrophy to auxotrophicmutants or resistance to heavy metals on wild-type strains. Expressioncontrol sequences for yeast vectors include promoters for glycolyticenzymes (Hess et al., J. Adv. Enzyme Reg. 1968, 7, 149), PHO5 geneencoding acid phosphatase, CUP1 gene, ARG3 gene, GAL genes promoters andsynthetic promoter sequences. Other control elements useful in yeastexpression are terminators and mRNA leader sequences. The 5′ codingsequence is particularly useful since it typically encodes a signalpeptide comprised of hydrophobic amino acids which direct the secretionof the protein from the cell. Suitable signal sequences can be encodedby genes for secreted yeast proteins such as the yeast invertase geneand the α-factor gene, acid phosphatase, killer toxin, the alpha-matingfactor gene and recently the heterologous inulinase signal sequencederived from INULA gene of Kluyveromyces marxianus. Suitable vectorshave been developed for expression in Pichia pastoris and Saccharomycescerevisiae.

A variety of P. pastoris expression vectors are available based onvarious inducible or constitutive promoters (Cereghino and Cregg, FEMSMicrobiol. Rev. 2000, 24:45-66). For the production of cytosolic andsecreted proteins, the most commonly used P. pastoris vectors containthe very strong and tightly regulated alcohol oxidase (AOX1) promoter.The vectors also contain the P. pastoris histidinol dehydrogenase (HIS4)gene for selection in his4 hosts. Secretion of foreign protein requirethe presence of a signal sequence and the S. cerevisiae prepro alphamating factor signal sequence has been widly and successfully used inPichia expression system. Expression vectors are integrated into the P.pastoris genome to maximize the stability of expression strains. As inS. cerevisiae, cleavage of a P. pastoris expression vector within asequence shared by the host genome (AOX1 or HIS4) stimulates homologousrecombination events that efficiently target integration of the vectorto that genomic locus. In general, a recombinant strain that containsmultiple integrated copies of an expression cassette can yield moreheterologous protein than single-copy strain. The most effective way toobtain high copy number transformants requires the transformation ofPichia recipient strain by the sphaeroplast technique (Cregg et all1985, Mol. Cell. Biol. 5: 3376-3385).

The preparation of the replicable expression vector may be carried outconventionally with appropriate enzymes for restriction, polymerisationand ligation of the DNA, by procedures described in, for example,Maniatis et al. cited above.

The recombinant host cell is prepared, in accordance with the invention,by transforming a host cell with a replicable expression vector of theinvention under transforming conditions. Suitable transformingconditions are conventional and are described in, for example, Maniatiset al. cited above, or “DNA Cloning” Vol. II, D. M. Glover ed., IRLPress Ltd, 1985.

The choice of transforming conditions depends upon the choice of thehost cell to be transformed. For example, in vivo transformation using alive viral vector as the transforming agent for the polynucleotides ofthe invention is described above. Bacterial transformation of a hostsuch as E. coli may be done by direct uptake of the polynucleotides(which may be expression vectors containing the desired sequence) afterthe host has been treated with a solution of CaCl₂ (Cohen et al., Proc.Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixtureof rubidium chloride (RbC1), MnCl₂, potassium acetate and glycerol, andthen with 3-[N-morpholino]-propane-sulphonic acid, RbC1 and glycerol orby electroporation. Transformation of lower eukaryotic organisms such asyeast cells in culture by direct uptake may be carried out for exampleby using the method of Hinnen et al (Proc. Natl. Acad. Sci. 1978,75:1929-1933). Mammalian cells in culture may be transformed using thecalcium phosphate co-precipitation of the vector DNA onto the cells(Graham & Van der Eb, Virology 1978, 52, 546). Other methods forintroduction of polynucleotides into mammalian cells include dextranmediated transfection, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) intoliposomes, and direct micro-injection of the polynucleotides intonuclei.

The invention also extends to a host cell transformed with a nucleicacid encoding the protein of the invention or a replicable expressionvector of the invention.

Culturing the transformed host cell under conditions permittingexpression of the DNA polymer is carried out conventionally, asdescribed in, for example, Maniatis et al. and “DNA Cloning” citedabove. Thus, preferably the cell is supplied with nutrient and culturedat a temperature below 50° C., preferably between 25° C. and 42° C.,more preferably between 25° C. and 35° C., most preferably at 30° C. Theincubation time may vary from a few minutes to a few hours, according tothe proportion of the polypeptide in the bacterial cell, as assessed bySDS-PAGE or Western blot.

The product may be recovered by conventional methods according to thehost cell and according to the localisation of the expression product(intracellular or secreted into the culture medium or into the cellperiplasm). Thus, where the host cell is bacterial, such as E. coli itmay, for example, be lysed physically, chemically or enzymatically andthe protein product isolated from the resulting lysate. Where the hostcell is mammalian, the product may generally be isolated from thenutrient medium or from cell free extracts. Where the host cell is ayeast such as Saccharomyces cerevisiae or Pichia pastoris, the productmay generally be isolated from from lysed cells or from the culturemedium, and then further purified using conventional techniques. Thespecificity of the expression system may be assessed by western blot orby ELISA using an antibody directed against the polypeptide of interest.

Conventional protein isolation techniques include selectiveprecipitation, adsorption chromatography, and affinity chromatographyincluding a monoclonal antibody affinity column. When the proteins ofthe present invention are expressed with a histidine tail (His tag),they can easily be purified by affinity chromatography using an ionmetal affinity chromatography column (IMAC) column. The metal ion, maybe any suitable ion for example zinc, nickel, iron, magnesium or copper,but is preferably zinc or nickel. Preferably the IMAC buffer containsdetergent, preferably an anionic detergent such as SDS, more preferablya non-ionic detergent such as Tween 80, or a zwitterionic detergent suchas Empigen BB, as this may result in lower levels of endotoxin in thefinal product.

Further chromatographic steps include for example a Q-Sepharose stepthat may be operated either before of after the IMAC column. Preferablythe pH is in the range of 7.5 to 10, more preferably from 7.5 to 9.5,optimally between 8 and 9.

The proteins of the invention can thus be purified according to thefollowing protocol. After cell disruption, cell extracts containing theprotein can be solubilised in a pH 8.5 Tris buffer containing urea (8.0M for example), and SDS (from 0.5% to 1% for example). Aftercentrifugation, the resulting supernatant may then be loaded onto on toan IMAC (Nickel) Sepharose FF column equilibrated with a pH 8.5 Trisbuffer. The column may then be washed with a high salt containing buffer(eg 0.75-1.5 m NaC1, 15 mM pH 8.5 Tris buffer). The column mayoptionally then be washed again with phosphate buffer without salt. Theproteins of the invention may be eluated from the column with animidazole-containing buffered solution. The proteins can then besubmitted to an additional chromatographic step, such as to an anionexchange chromatography (Q Sepharose for example).

The proteins of the present invention are provided either soluble in aliquid form or in a lyophilised form, which is the preferred form. It isgenerally expected that each human dose will comprise 1 to 1000 μg ofprotein, and preferably 30-300 μg. The purification process can alsoinclude a carboxyamidation step whereby the protein is first reduced inthe presence of Glutathion and then carboxymethylated in the presence ofiodoacetamide. This step offers the advantage of controling theoxidative aggregation of the molecule with itself or with host cellprotein contaminants through covalent bridging with disulphide bonds.

The present invention also provides pharmaceutical and immunogeniccompositions comprising a protein of the present invention in apharmaceutically acceptable excipient. A preferred vaccine compositioncomprises at least a protein according to the invention. Said proteinhas, preferably, blocked thiol groups and is highly purified, e.g. hasless than 5% host cell contamination. Such vaccine may optionallycontain one or more other tumour-associated antigen and derivatives. Forexample, suitable other associated antigen include prostase, PAP-1, PSA(prostate specific antigen), PSMA (prostate-specific membrane antigen),PSCA (Prostate Stem Cell Antigen), STEAP.

In another embodiment, illustrative immunogenic compositions, such asfor example vaccine compositions, of the present invention comprise DNAencoding one or more of the fusion polypeptides as described above, suchthat the fusion polypeptide is generated in situ. As noted above, thepolynucleotide may be administered within any of a variety of deliverysystems known to those of ordinary skill in the art. Indeed, numerousgene delivery techniques are well known in the art, such as thosedescribed by Rolland, Crit. Rev. Therap. Drug Carrier Systems15:143-198, 1998, and references cited therein. Appropriatepolynucleotide expression systems will, of course, contain the necessaryregulatory DNA regulatory sequences for expression in a patient (such asa suitable promoter and terminating signal). Alternatively, bacterialdelivery systems may involve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

Therefore, in certain embodiments, polynucleotides encoding immunogenicpolypeptides described herein are introduced into suitable mammalianhost cells for expression using any of a number of known viral-basedsystems. In one illustrative embodiment, retroviruses provide aconvenient and effective platform for gene delivery systems. A selectednucleotide sequence encoding a polypeptide of the present invention canbe inserted into a vector and packaged in retroviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to a subject. A number of illustrative retroviral systemshave been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman(1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin(1993) Cur. Opin. Genet. Develop. 3:102-109.

In addition, a number of illustrative adenovirus-based systems have alsobeen described. Unlike retroviruses which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476). Since humans are sometimes infected bycommon human adenovirus serotypes such as AdHu5, a significantproportion of the population have a neutralizing antibody response tothe adenovirus, which is likley to effect the immune response to aheterologous antigen in a recombinant vaccine based system. Non-humanprimate adenoviral vectors such as the chimpanzee adenovirus 68 (AdC68,Fitzgerald et al. (2003) J. Immunol 170(3):1416-22)) are may offer analternative adenoviral system without the disadvantage of a pre-existingneutralising antibody response.

Various adeno-associated virus (MV) vector systems have also beendeveloped for polynucleotide delivery. MV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Additional viral vectors useful for delivering the nucleic acidmolecules encoding polypeptides of the present invention by genetransfer include those derived from the pox family of viruses, such asvaccinia virus and avian poxvirus. By way of example, vaccinia virusrecombinants expressing the novel molecules can be constructed asfollows. The DNA encoding a polypeptide is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the polypeptideof interest into the viral genome. The resulting TK.sup.(−) recombinantcan be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently usedto provide for inducible, transient expression or coexpression of one ormore polypeptides described herein in host cells of an organism. In thisparticular system, cells are first infected in vitro with a vacciniavirus recombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide or polynucleotides of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into polypeptide by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used for delivery ofpolynucleotide compositions of the present invention, such as thosevectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE)can also be used, illustrative examples of which can be found in U.S.Pat. Nos. 5,505,947 and 5,643,576.

The compositions of the present invention can be delivered by a numberof routes such as intramuscularly, subcutaneously, intraperitonally orintravenously.

In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells. In a preferred embodiment, the composition is deliveredintradermally. In particular, the composition is delivered by means of agene gun (particularly particle bombardment) administration techniqueswhich involve coating the vector on to a bead (eg gold) which are thenadministered under high pressure into the epidermis; such as, forexample, as described in Haynes et al, J Biotechnology 44: 37-42 (1996).

In one illustrative example, gas-driven particle acceleration can beachieved with devices such as those manufactured by PowderjectPharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison,Wis.), some examples of which are described in U.S. Pat. Nos. 5,846,796;6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799. Thisapproach offers a needle-free delivery approach wherein a dry powderformulation of microscopic particles, such as polynucleotide, areaccelerated to high speed within a helium gas jet generated by a handheld device, propelling the particles into a target tissue of interest,typically the skin. The particles are preferably gold beads of a 0.4-4.0μm, more preferably 0.6-2.0 μm diameter and the DNA conjugate coatedonto these and then encased in a cartridge or cassette for placing intothe “gene gun”.

In a related embodiment, other devices and methods that may be usefulfor gas-driven needle-less injection of compositions of the presentinvention include those provided by Bioject, Inc. (Portland, Oreg.),some examples of which are described in U.S. Pat. Nos. 4,790,824;5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

It is possible for the immunogen component comprising the nucleotidesequence encoding the antigenic peptide, to be administered on a onceoff basis or to be administered repeatedly, for example, between 1 and 7times, preferably between 1 and 4 times, at intervals between about 1day and about 18 months. However, this treatment regime will besignificantly varied depending upon the size of the patient, the diseasewhich is being treated/protected against, the amount of nucleotidesequence administered, the route of administration, and other factorswhich would be apparent to a skilled medical practitioner.

It is therefore another aspect of the present invention to provide forthe use of a protein or a DNA encoding said protein, as describedherein, in the manufacture of an immunogenic composition for elicitingan immune response in a patient. Preferably the immune response is to beelicited by sequential administration of i) the said protein followed bythe said DNA sequence; or ii) the said DNA sequence followed by the saidprotein. More preferably the DNA sequence is coated onto biodegradablebeads or delivered via a particle bombardment approach. Still morepreferably the protein ios adjuvanted, preferably with a TH-1 inducingadjuvant, preferably with a CpG/QS21 based adjuvant formulation.

The vectors which comprise the nucleotide sequences encoding antigenicpeptides are administered in such amount as will be prophylactically ortherapeutically effective. The quantity to be administered, is generallyin the range of one picogram to 16 milligram, preferably 1 picogram to10 micrograms for particle-mediated delivery, and 10 micrograms to 16milligram for other routes of nucleotide per dose. The exact quantitymay vary considerably depending on the weight of the patient beingimmunised and the route of administration.

Suitable techniques for introducing the naked polynucleotide or vectorinto a patient also include topical application with an appropriatevehicle. The nucleic acid may be administered topically to the skin, orto mucosal surfaces for example by intranasal, oral, intravaginal orintrarectal administration. The naked polynucleotide or vector may bepresent together with a pharmaceutically acceptable excipient, such asphosphate buffered saline (PBS). DNA uptake may be further facilitatedby use of facilitating agents such as bupivacaine, either separately orincluded in the DNA formulation. Other methods of administering thenucleic acid directly to a recipient include ultrasound, electricalstimulation, electroporation and microseeding which is described in U.S.Pat. No. 5,697,901.

Uptake of nucleic acid constructs may be enhanced by several knowntransfection techniques, for example those including the use oftransfection agents. Examples of these agents includes cationic agents,for example, calcium phosphate and DEAE-Dextran and lipofectants, forexample, lipofectam and transfectam. The dosage of the nucleic acid tobe administered can be altered.

The fusion proteins and encoding polypeptides according to the inventioncan also be formulated as a pharmaceutical/immunogenic composition, e.g.as a vaccine. Accordingly therefore, the present invention also providesfor a pharmaceutical/immunogenic composition comprising a fusion proteinof the present invention in a pharmaceutically acceptable excipient.Accordingly there is also provided a process for the preparation of animmunogenic composition according to the present invention, comprisingadmixing the fusion protein of the invention or the encodingpolynucleotide with a suitable adjuvant, diluent or otherpharmaceutically acceptable carrier.

The fusion proteins of the present invention are provided preferably atleast 80% pure more preferably 90% pure as visualised by SDS PAGE.Preferably the proteins appear as a single band by SDS PAGE.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds. Powell M. F. & Newman M. J). (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

The fusion proteins of the present invention and encodingpolynucleotides are preferably adjuvanted in the vaccine formulation ofthe invention. Certain adjuvants are commercially available as, forexample, Freund's Incomplete Adjuvant and Complete Adjuvant (DifcoLaboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatised polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol 7:145-173, 1989.

Preferred TH-1 inducing adjuvants are selected from the group ofadjuvants comprising: 3D-MPL, QS21, a mixture of QS21 and cholesterol,and a CpG oligonucleotide or a mixture of two or more said adjuvants.Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from Corixa Corporation(Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as CarbopoIR toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 as disclosed in WO 00/09159 and in WO00/62800. Preferably the formulation additionally comprises an oil inwater emulsion and tocopherol.

In a yet further embodiment the present invention provides animmunogenic composition comprising a fusion protein according to theinvention, and further comprising D3-MPL, a saponin preferably QS21 anda CpG oligonucleotide, optionally formulated in an oil in wateremulsion.

Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformula (I): HO(CH₂CH₂O)_(n)-A-R, wherein, n is 1-50, A is a bond or—C(O)—, R is C₁₋₅₀ alkyl or Phenyl C₁₋₅₀ alkyl. One embodiment of thepresent invention consists of a vaccine formulation comprising apolyoxyethylene ether of general formula (I), wherein n is between 1 and50, preferably 4-24, most preferably 9; the R component is C₁₋₁₅₀,preferably C₄-C₂₀ alkyl and most preferably C₁₋₂ alkyl, and A is a bond.The concentration of the polyoxyethylene ethers should be in the range0.1-20%, preferably from 0.1-10%, and most preferably in the range0.1-1%. Preferred polyoxyethylene ethers are selected from the followinggroup: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether,polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.Polyoxyethylene ethers such as polyoxyethylene lauryl ether aredescribed in the Merck index (12^(th) edition: entry 7717). Theseadjuvant molecules are described in WO 99/52549. The polyoxyethyleneether according to the general formula (I) above may, if desired, becombined with another adjuvant. For example, a preferred adjuvantcombination is preferably with CpG as described in the pending UK patentapplication GB 9820956.2.

It is an embodiment of the invention that the antigens, includingnucleic acid vector, of the invention be utilised with immunostimulatoryagent. Preferably the immunostimulatory agent is administered at thesame time as the antigens of the invention and in preferred embodimentsare formulated together. It is another embodiment of the invention thatthe antigen and immunostimulatory agent (or vice versa) are administeredsequentially to the same or adjacent sites, separated in time by periodsof between 0-100 hours. Such immunostimulatory agents include but arenot limited to: synthetic imidazoquinolines such as imiquimod [S-26308,R-837], (Harrison, et al., Vaccine 19: 1820-1826, 2001; and resiquimod[S-28463, R-848] (Vasilakos, et al., Cellular immunology 204: 64-74,2000.; Schiff bases of carbonyls and amines that are constitutivelyexpressed on antigen presenting cell and T-cell surfaces, such astucaresol (Rhodes, J. et al., Nature 377: 71-75, 1995), cytokine,chemokine and co-stimulatory molecules as either protein or peptide,including for example pro-inflammatory cytokines such as Interferon,GM-CSF, IL-1 alpha, IL-1 beta, TGF-alpha and TGF-beta, Th1 inducers suchas interferon gamma, IL-2, IL-12, IL-15, IL-18 and IL-21, Th2 inducerssuch as IL-4, IL-5, IL-6, IL-10 and IL-13 and other chemokine andco-stimulatory genes such as MCP-1, MIP-1 alpha, MIP-1 beta, RANTES,TCA-3, CD80, CD86 and CD40L, other immunostimulatory targeting ligandssuch as CTLA-4 and L-selectin, apoptosis stimulating proteins andpeptides such as Fas, (49), synthetic lipid based adjuvants, such asvaxfectin, (Reyes et al., Vaccine 19: 3778-3786, 2001) squalene,alpha-tocopherol, polysorbate 80, DOPC and cholesterol, endotoxin,[LPS], (Beutler, B., Current Opinion in Microbiology 3: 23-30, 2000);CpG oligo- and di-nucleotides (Sato, Y. et al., Science 273 (5273):352-354, 1996; Hemmi, H. et al., Nature 408: 740-745, 2000) and otherpotential ligands that trigger Toll receptors to produce Th1-inducingcytokines, such as synthetic Mycobacterial lipoproteins, Mycobacterialprotein p19, peptidoglycan, teichoic acid and lipid A.

Other suitable adjuvant include CT (cholera toxin, subunites A and B)and LT (heat labile enterotoxin from E. coli, subunites A and B), heatshock protein family (HSPs), and LLO (listeriolysin O; WO 01/72329).

Where the immunostimulatory agent is a protein, the agent may beadministered either as a protein or as a polynucleotide encoding theprotein.

Other suitable delivery systems include microspheres wherein theantigenic material is incorporated into or conjugated to biodegradablepolymers/microspheres so that the antigenic material can be mixed with asuitable pharmaceutical carrier and used as a vaccine. The term“microspheres” is generally employed to describe colloidal particleswhich are substantially spherical and have a diameter in the range 10 nmto 2 mm. Microspheres made from a very wide range of natural andsynthetic polymers have found use in a variety of biomedicalapplications. This delivery system is especially advantageous forproteins having short half-lives in vivo requiring multiple treatmentsto provide efficacy, or being unstable in biological fluids or not fullyabsorbed from the gastrointestinal tract because of their relativelyhigh molecular weights. Several polymers have been described as a matrixfor protein release. Suitable polymers include gelatin, collagen,alginates, dextran. Preferred delivery systems include biodegradablepoly(DL-lactic acid) (PLA), poly(lactide-co-glycolide) (PLG),poly(glycolic acid) (PGA), poly(ε-caprolactone) (PCL), and copolymerspoly(DL-lactc-co-glycolic acid) (PLGA). Other preferred systems includeheterogeneous hydrogels such as poly(ether ester) multiblock copolymers,containing repeating blocks based on hydrophilic poly-(ethylene glycol)(PEG) and hydrophobic poly(butylene terephtalate) (PBT), orpoly(ehtykene glycol)-terephtalate/poly(-butylene terephtalate)(PEGT/PBT) (Sohier et al. Eur. J. Pharm and Biopharm, 2003, 55,221-228). Systems are preferred which provide a sustained release for 1to 3 months such as PLGA, PLA and PEGT/PBT.

It is possible for the immunogenic or vaccine composition to beadministered on a once off basis or, preferably, to be administeredrepeatedly, as many times as necessary, for example, between 1 and 7times, preferably between 1 and 4 times, at intervals between about 1day and about 18 months, preferably one month. This may be optionallyfollowed by dosing at regular intervals of between 1 and 12 months for aperiod up to the remainder of the patient's life. In a preferredembodiment the patient receives the antigen in different forms in a“prime boost” regime. Thus for example the antigen, the fusion protein,is first administered as a protein adjuvant base formulation and thensubsequently administered as a DNA based vaccine. This administrationmode is preferred. The preferred adjuvant is a combination of aCpG-containing oligonucleotide and a saponin derivative, particularlythe combination of CpG and QS21 as disclosed in WO 00/09159 and in WO00/62800. The uptake of naked DNA may be increased by coating the DNAonto biodegradable beads, which are efficiently transported into thecells. Alternatively the DNA can be delivered via a particle bombardmentapproach, for example, gas-driven particle acceleration with devicessuch as those manufactured by Powderject Pharmaceuticals PLC (Oxford,UK) and Powderject Vaccines Inc. (Madison, Wis.) as taught herein. Thisapproach offers a needle-free delivery approach wherein a dry powderformulation of microscopic particles, such as polynucleotide orpolypeptide particles, are accelerated to high speed within a helium gasjet generated by a hand held device, propelling the particles into atarget tissue of interest.

In another preferred embodiment, the DNA based vaccine will beadministered first, followed by the protein adjuvant base formulation.Still another embodiment will concern the delivery of the DNA constructby means of specialised delivery vectors, preferably by the means ofviral system, most preferably by the means of adenoviral-based systems.Other suitable viral-based systems of DNA delivery include retroviral,lentiviral, adeno-associated viral, herpes viral and vaccinia-viralbased systems.

In another preferred embodiment, the protein adjuvant base formulationand DNA based vaccine may be co-administered at adjacent or overlappingsites. Dependent upon the nature of the DNA vaccine formulation, thiscan be achieved by mixing the DNA and protein adjuvant formulationsprior to administration or by simultaneously administration of the DNAand protein adjuvant formulation.

The treatment regime will be significantly varied depending upon thesize and species of patient concerned, the amount of nucleic acidvaccine and/or protein composition administered, the route ofadministration, the potency and dose of any adjuvant compounds used andother factors which would be apparent to a skilled medical practitioner.

Within further aspects, the present invention provides methods forstimulating an immune response in a patient, preferably a T cellresponse in a human patient, comprising administering a pharmaceuticalcomposition described herein. The patient may be afflicted with lung orcolon cancer or colorectal cancer or breast cancer, in which case themethods provide treatment for the disease, or patient considered at riskfor such a disease may be treated prophylactically.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient a pharmaceutical composition as recitedabove. The patient may be afflicted with, for example, sarcoma,prostate, ovarian, bladder, lung, colon, colorectal or breast cancer, inwhich case the methods provide treatment for the disease, or patientconsidered at risk for such a disease may be treated prophylactically.

The present invention further provides, within other aspects, methodsfor removing tumour cells from a biological sample, comprisingcontacting a biological sample with T cells that specifically react witha polypeptide of the present invention, wherein the step of contactingis performed under conditions and for a time sufficient to permit theremoval of cells expressing the protein from the sample.

Within related aspects, methods are provided for inhibiting thedevelopment of a cancer in a patient, comprising administering to apatient a biological sample treated as described above.

Methods are further provided, within other aspects, for stimulatingand/or expanding T cells specific for a polypeptide of the presentinvention, comprising contacting T cells with one or more of: (i) apolypeptide as described above; (ii) a polynucleotide encoding such apolypeptide; and/or (iii) an antigen presenting cell that expresses sucha polypeptide; under conditions and for a time sufficient to permit thestimulation and/or expansion of T cells. Isolated T cell populationscomprising T cells prepared as described above are also provided.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient an effective amount of a T cell population asdescribed above.

The present invention further provides methods for inhibiting thedevelopment of a cancer in a patient, comprising the steps of: (a)incubating CD4+ and/or CD8+ T cells isolated from a patient with one ormore of: (i) a polypeptide disclosed herein; (ii) a polynucleotideencoding such a polypeptide; and (iii) an antigen-presenting cell thatexpressed such a polypeptide; and (b) administering to the patient aneffective amount of the proliferated T cells, and thereby inhibiting thedevelopment of a cancer in the patient. Proliferated cells may, but neednot, be cloned prior to administration to the patient.

According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaïve T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide of the invention(or portion or other variant thereof) such that the encoded polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

Definitions

Also provided by the invention are methods for the analysis of charactersequences or strings, particularly genetic sequences or encoded proteinsequences. Preferred methods of sequence analysis include, for example,methods of sequence homology analysis, such as identity and similarityanalysis, DNA, RNA and protein structure analysis, sequence assembly,cladistic analysis, sequence motif analysis, open reading framedetermination, nucleic acid base calling, codon usage analysis, nucleicacid base trimming, and sequencing chromatogram peak analysis.

A computer based method is provided for performing homologyidentification. This method comprises the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and comparing said firstpolynucleotide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology. A computer based method isalso provided for performing homology identification, said methodcomprising the steps of: providing a first polypeptide sequencecomprising the sequence of a polypeptide of the invention in a computerreadable medium; and comparing said first polypeptide sequence to atleast one second polynucleotide or polypeptide sequence to identifyhomology.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Infommatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heine, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GAP program in the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), and FASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA85; 2444-2448 (1988). The BLAST family of programs is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

Parameters for polypeptide sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Henikoff and Henikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 8

Gap Length Penalty: 2

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to any of the referencesequences of SEQ ID NO:9 to SEQ ID NO:16, wherein said polynucleotidesequence may be identical to any the reference sequences of SEQ ID NO:9to SEQ ID NO:16 or may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in any of SEQ ID NO:9 to SEQ ID NO:16 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in any ofSEQ ID NO:9 to SEQ ID NO:16, or:n _(n) ≦x _(n)−(x _(n) ·y)wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in any of SEQ ID NO:9 to SEQ ID NO:16, y is0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%,0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and · is thesymbol for the multiplication operator, and wherein any non-integerproduct of x_(n) and y is rounded down to the nearest integer prior tosubtracting it from x_(n). Alterations of polynucleotide sequencesencoding the polypeptides of any of SEQ ID NO:1 to SEQ ID NO:8 maycreate nonsense, missense or frameshift mutations in this codingsequence and thereby alter the polypeptide encoded by the polynucleotidefollowing such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to any of the reference sequences of SEQ ID NO:9 to SEQID NO:16, that is it may be 100% identical, or it may include up to acertain integer number of nucleic acid alterations as compared to thereference sequence such that the percent identity is less than 100%identity. Such alterations are selected from the group consisting of atleast one nucleic acid deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference polynucleotide sequenceor anywhere between those terminal positions, interspersed eitherindividually among the nucleic acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleic acid alterations for a given percent identity is determined bymultiplying the total number of nucleic acids in any of SEQ ID NO:9 toSEQ ID NO:16 by the integer defining the percent identity divided by 100and then subtracting that product from said total number of nucleicacids in any of SEQ ID NO:9 to SEQ ID NO:16, orn _(n) ≦x _(n)−(x _(n) ·y),wherein n_(n) is the number of nucleic acid alterations, x_(n) is thetotal number of nucleic acids in any of SEQ ID NO:9 to SEQ ID NO:16, yis, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., · is thesymbol for the multiplication operator, and wherein any non-integerproduct of x_(n) and y is rounded down to the nearest integer prior tosubtracting it from x_(n).(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95,97 or 100% identity to the polypeptide reference sequence of any of SEQID NO:1 to SEQ ID NO:8, wherein said polypeptide sequence may beidentical to any of the reference sequence of SEQ ID NO:1 to SEQ ID NO:8or may include up to a certain integer number of amino acid alterationsas compared to the reference sequence, wherein said alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of amino acid alterations is determined by multiplying thetotal number of amino acids in any of SEQ ID NO:1 to SEQ ID NO:8 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in any ofSEQ ID NO:1 to SEQ ID NO:8, or:n _(a) ≦x _(a)−(x _(a) ·y),wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of any of SEQ ID NO:1 to SEQ IDNO:8, that is it may be 100% identical, or it may include up to acertain integer number of amino acid alterations as compared to thereference sequence such that the percent identity is less than 100%identity. Such alterations are selected from the group consisting of atleast one amino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence. The number of amino acid alterations for a given %identity is determined by multiplying the total number of amino acids inany of SEQ ID NO:1 to SEQ ID NO:8 by the integer defining the percentidentity divided by 100 and then subtracting that product from saidtotal number of amino acids in any of SEQ ID NO:1 to SEQ ID NO:8, or:n _(a) ≦x _(a)−(x_(a) ·y)wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in any of SEQ ID NO:1 to SEQ ID NO:8, y is,for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and · is thesymbol for the multiplication operator, and wherein any non-integerproduct of x_(a) and y is rounded down to the nearest integer prior tosubtracting it from x_(a).

FIGURE LEGENDS

FIG. 1: Sequence information for C-LytA. Each repeat has been defined onthe basis of both multiple sequence alignment and secondary structureprediction using the following alignment programs: 1) MatchBox(Depiereux E et al. (1992) Comput Applic Biosci 8:501-9); 2) ClustalW(Thompson J D et al. (1994) Nucl Acid Res 22:4673-80); 3) Block-Maker(Henikoff S et al (1995) Gene 163:gc17-26)

FIG. 2: CPC and native Constructs (SEQ ID NOs. 27-36)

FIG. 3: Schematic structure of CPC-p501 His fusion protein expressed inS. cerevisiae

FIG. 4: Primary structure of CPC-P501 His fusion protein (SEQ ID NO.41)

FIG. 5: Nucleotide sequence of CPC P501 His (pRIT15201) (SEQ ID NO.42)

FIG. 6: Cloning strategy for generation of plasmid pRIT 15201

FIG. 7: Plasmid map of pRIT15201

FIG. 8. Comparative expression of CPC P501 and P501 in S. cerevisiaestrain DC5

FIG. 9: Production of CPC-P501S HIS (Y1796) at small scale. FIG. 9Arepresents the antigen productivity as estimated by SDS-PAGE with silverstaining; FIG. 9B represents the antigen productivity as estimated bywestern blot.

FIG. 10: Purification scheme of CPC-P501-His produced by Y1796.

FIG. 11: Pattern of CPC P501 His purified protein (4-12% Novex Nu-Pagepolyacrylamide precasted gels).

FIG. 12: Native full-length P501S sequence (SEQ ID NO:17)

FIG. 13: Sequence of the CPC-P501S expression cassette of JNW735 (SEQ IDNO:18)

FIG. 14: Two codon optimised P501S sequences (SEQ ID NO:19-20)

FIG. 15: Re-engineered codon optimised sequence 19 (SEQ ID NO:21)

FIG. 16: Re-engineered codon optimised sequence 20 (SEQ ID NO:22)

FIG. 17: The starting sequence for the optimisation of CPC (SEQ IDNO:23)

FIG. 18: Representative codon optimised CPC sequences (SEQ ID NO:24-25)

FIG. 19: Engineered CPC codon optimised sequence (SEQ ID NO:26)

FIG. 20: P501S CPC fusion candidate constructs and sequences (SEQ IDNOs. 3740 & 4548)

FIG. 21: Western blot analysis of CHO cells following transienttransfection with P501S (JNW680), CPC-P501S (JNW735) and empty vectorcontrol.

FIG. 22: Anti-P501S antibody responses following immunisation at day 0,21 & 42 with pVAC-P501S (JNW680, mice B1-9) or Empty vector (pVAC, miceA1-6). A pre-bleed was taken at day −1. Subsequently bleeds were takenat day 28 and day 49 (mice A1-3, B1-3) and day 56 (mice A4-6, B4-9). Allsera was tested at 1/100 dilution. The results for the pVAC immunisedmice were averaged. The results for the individual pVAC-P501S immunisedmice are shown. As a positive control, sera from Adeno-P501S immunisedmice (Corixa Corp, diluted 1/100) is included.

FIG. 23: Peptide library screen using C57BL/6 mice immunised at day 0,21, 42, and 70 with pVAC-P501S (JNW680). All peptides were used at afinal concentration of 50 μg/ml. Peptides 1-50 are overlapping 15-20mersobtained from Corixa. Peptides 51-70 are predicted 8-9mer Kb and Dbepitopes and were ordered from Mimotopes (UK). Samples 71-72 and 73-78are DMSO controls and no peptide controls respectively. Graph A showsthe IFN-γ responses whilst Graph B shows the IL-2 responses. Peptidesselected for use in subsequent immunoassays are shown in black.

FIG. 24: Cellular responses by ELISPOT at day 77 following PMIDimmunisation at day 0, 21, 42, and 70 with pVAC-P501S (JNW680, B6-9) andpVAC empty (A4-6). Peptide 18, 22 & 48 were used at 50 μg/ml. CPC-P501Sprotein was used at 20 μg/ml. Graph A shows the IFN-γ responses whilstGraph B shows the IL-2 responses.

FIG. 25: Comparison of P501S and CPC-P501S. Cellular responses weremeasured by IL-2 ELISPOT using peptide 22 (10 μg/ml) at day 28. Micewere immunised by PMID at day 0 and 21 with pVAC empty (control),pVAC-P501S (JNW680) and CPC-P501S (JNW735).

FIG. 26: Immune response (lymphoproliferation on spleen cells) followingprotein immunisation with CPC-P501S.

FIG. 27: Evaluation of the immune response to different CPC-P501Sconstructs. Cellular responses were measured by IL-2 ELISPOT at day 28.Mice were immunised by PMID at day 0 and 21 with p7313-ie empty(control), JNW735 and CPC-P501S constructs (JNW770, 771 and 773)

FIG. 28: MUC-1 CPC sequences (SEQ ID NOs. 49 & 50)

FIG. 29: ss-CPC-MUC-1 sequences (SEQ ID NOs. 51 & 52)

The invention will be further described by reference to the followingexamples:

EXAMPLE I Preparation of the Recombinant Yeast Strain Y1796 ExpressingP501 Fusion Protein Containing a C-LytA-P2-C-LytA (CPC) as fusionpartner

1.—Protein Design

The structure of the fusion protein C-P2-C-p501 (alternatively namedCPC-P501) to be expressed in S. cerevisiae is depicted in FIG. 3. Thisfusion contains the C-terminal region of gene LytA (residues 187 to306), in which the P2 fragment of tetanus toxin (residues 830-843) hasbeen inserted. The P2 fragment is placed between the residues 277 and278 of C-Lyt-A. The C-lytA fragment containing the P2 insertion isfollowed by P501 (residues amino acid 51 to 553) and by the His tail.

The primary structure of the resulting fusion protein has the sequencedescribed in FIG. 4 and the coding sequence corresponding to the aboveprotein design is in FIG. 5.

2.—Cloning Strategy for the Generation of a Yeast Plasmid ExpressingCPC-P501 (51-553)-His Fusion Protein

-   -   The starting material is the yeast vector pRIT15068 (UK patent        application 0015619.0).    -   This vector contains the yeast Cup1 promoter, the yeast alpha        prepro signal coding sequence and the coding sequence        corresponding to residues 55 to 553 of P501S followed by His        tail.    -   The cloning strategy outlined in FIG. 6 include the following        steps:        a) The first step is the insertion of P2 sequence        (codon-optimised for yeast expression) in frame, inside the        C-lytA coding sequence. The C-lytA coding sequence is harbored        by plasmid pRIT 14662 (PCT/EP99100660). The insertion is done        using an adaptor formed by two complementary oligonucleotides        named P21 and P22 into the plasmid pRIT 14662 previously open by        NcoI

The sequence of P21 and P22 is:

P21 5′ catgcaatacatcaaggctaactctaagttcattggtatcactgaaggcgt 3′

P22 3′ gttatgtagttccgattgagattcaagtaaccatagtgacttccgcagtac 5′

After ligation and transformation of E. coli and transformantcharacterization, the plasmid named pRIT15199 is obtained.

b) The second step is the preparation of C-lytA-P2-C-lytA DNA fragmentby PCR amplification. The amplification is performed using pRIT15199 astemplate and the oligonucleotides named C-LytANOTATG and C-LytA-aa55.The sequence of both oligonucleotides being:

C-LytANOTATG

=5′aaaaccatggcggccgcttacgtacattccgacggctcttatccaaaagacaag 3′C-LytA-aa55=5′aaacatgtacatgaacttttctggcctgtctgccagtgttc 3′

The amplified fragment is treated with the restriction enzymes NcoI andAfl III to generate the respective cohesive ends.

c) The next step is the ligation of the above fragment with vectorpRIT15068 (largest fragment obtained after NcoI treatment) to generatethe complete fusion protein coding sequence. After ligation and E. colitransformation the plasmid named pRIT15200 is obtained. In this plasmidthe remaining unique NcoI site contains the ATG coding for the startcodon.

d) In the next step a NcoI fragment containing the CUP1 promoter and aportion of 2μ plasmid sequences is prepared from plasmid PRIT 15202.Plasmid pRIT 15202 is a yeast 2μ derivative containing the CUP1 promoterwith an NcoI site at ATG (ATG sequence: AAACC ATG)

e) The NcoI fragment isolated from PRIT 15202 is ligated to pRIT15200,previously open with NcoI, in the righ orientation, in such a way thepCUP1 promoter is at the 5′ side of the coding sequence. This results inthe generation of a final expression plasmid named pRIT15201 (see FIG.7).

3.—Preparation of the Recombinant Yeast Strain Y1796 (RIX4440)

The plasmid pRIT 15201 is used to transform the S. cerevisiae strain DC5(ATCC 20820). After selection and characterisation of the yeasttransformants containing the plasmid pRIT 15201 a recombinant yeaststrain named Y1796 expressing CPC-P501-His fusion protein is obtained.The protein after reduction and carboxyamidation, is isolated andpurified by affinity chromatography (IMAC) followed by anion exchangechromatography (Q Sepharose FF).

EXAMPLE II

In analogous fashion proteins constructs as depicted in FIG. 2 may beexpressed utilising the corresponding DNA sequences shown therein. Inparticular, yeast strain SC333 (construct 2) corresponds to Y1796 strainbut expressing P501₅₅₋₅₅₃ devoid of the CPC fusion partner. Yeast strainY1800 (construct 3) corresponds to Y1796 strain but additionallycomprises the native sequence signal for P501S (aa1-aa34), while yeaststrain Y1802 (construct 4) comprises the alpha pre signal sequenceupstream CPC-P501S sequence. Yeast strain Y1790 (construct 5) isexpressing a P501S construct devoid of CPC and having the alpha preprosignal sequence.

EXAMPLE III Preparation of Purified CPC-P501

1.—Production of CPC-P501S HIS (Y1796) at Small Scale

For Y1796, in minimal medium supplemented with histidine, expression isinduced in log phase by addition of CuSO4 ranging from 100 to 500 μM,and culture is maintained at 30°. Cells are harvested after 8 or 24Hinduction. Copper is added just before use and not mixed with medium inadvance.

For SDS PAGE analysis, yeast cells extraction is performed in citratephosphate buffer pH4.0+130 mM NaCl. Extraction is performed with glassbeads for small cell quantity and with French press for higher cellsquantity, and then mixed with sample buffer and SDS-PAGE analysed.Results of comparative analysis on SDS PAGE of the different constructsare depicted in FIG. 8 and summariosed in Table 2 below.

As shown in Table 1 below, the level of expression of the culture ismuch higher for Y1796 strain as compared to the expression level ofparent strain SC333, a strain expressing the corresponding P501S-Hisdevoid of CPC partner. Likewise, the presence of a signal sequence(alpha pre) does not affect the results discussed above: the level ofexpression of the culture is much higher for Y1802 strain as compared tothe expression level of corresponding strain Y1790, a strain expressingthe corresponding P501S-His devoid of CPC partner. TABLE 2 RecombinantSignal Fusion P501 aa Expression Strain Plasmid Promotor sequencePartner sequences level SC333 Ma333 CUP 1 — — 55-553-His ⊖ND Y1796 pRIT15201 CUP 1 — CPC 51-553-His +++ Y1802 pRIT 15219 CUP 1 α pre CPC51-553-His ++++ Y1790 pRIT 15068 CUP 1 α prepro — 55-553-His +CPC = clyta P2 clytaND = not detectable, even in western blot+ = detectable in western blot+++/++++ = detectable in western blot and visible in silver stained gels2.—Fermentation of Y1796 (RIX4440) at Larger Scale

100 μl of the working seed are spread on solid medium and grown forapproximately 24 h at 30° C. This solid pre-culture is then used toinoculate a liquid pre-culture in shake flasks.

This liquid pre-culture is grown for 20 h at 30° C. and transferred intoa 20 L fermenter. The fed-batch fermentation includes a growth phase ofabout 44 h and an induction phase of about 22 h.

The carbon source (glucose) was supplemented to the culture by acontinuous feeding. The residual glucose concentration was maintainedvery low (≦50 mg/L) in order to minimise the ethanol production byfermentation. This was realised by limiting the development of themicro-organism by limited glucose feed rate.

At the end of the growth phase, CUP1 promoter is induced by adding CuSO4in order to produce the antigen.

The absence of contaminations was checked by inoculating 10⁶ cells intostandard TSB and THI vials supplemented with nystatine and incubatedrespectively for 14 days at 20-25° C. and at 30-35° C. No growth wasobserved as expected.

3.—Antigen Characterisation and Productivity

Cell homogenates were prepared by French pressing of fermentationsamples harvested at different times during the induction phase andanalysed by SDS-PAGE and Western Blot. It was shown that the major partof the protein of interest was located in the insoluble fractionobtained from the cell homogenate after centrifugation. The SDS-PAGE andWestern Blot analyses shown in the Figures below were realised on thepellets obtained after centrifugation of these cell homogenates.

FIGS. 8 A and B show a kinetics of the antigen production during theinduction phase for culture PRO127. It appears that no antigenexpression occurred during the growth phase. The specific antigenproductivity seems to increase from the beginning of the induction phaseup to 6 h and then remained quite stable up to the end. But thevolumetric productivity increased by a factor 1.5 to 2 due to biomassaccumulation observed during the same period of time. The antigenproductivity was estimated at about 500 mg per litre of fermentationbroth by comparing purified reference of the antigen and crude extractson SDS-PAGE with silver staining (FIG. 9A) and WB analyses using ananti-P501S antibody (a murine ascite directed against P501S aa439-aa459used at a dilution of 1/1000) (FIG. 9B).

EXAMPLE IV Purification of CPC-P501 (51-553)-His Fusion Protein Producedby Y1796

After the cell breakage, the protein is associated with the pelletfraction. A carbamido-methylation of the molecule has been introduced inthe process in order to cope with the oxidative aggregation of themolecule with itself or with host cell protein contaminants throughcovalent bridging with disulphide bonds. The use of detergents has alsobeen required to manage the hydrophobic character of this protein (12trans-membrane domains predicted).

The purification protocol, developed for the scale of 1 L of culture OD(optical density) 120, is described in FIG. 10. All the operations areperformed at room temperature (RT).

According to DOC TCA BCA protein assay, the global purification yield is30-70 mg of purified antigen/L of culture OD 120. The yield is linked tothe level of expression of the culture and is higher as compared to thepurification yield of parent strain expressing unfused P501S-His.

The protein assay is performed as followed: proteins are firstprecipitated using TCA (trichloroacetic acid) in the presence of DOC(deoxycholate) then dissolved in a alcaline medium in the presence ofSDS. The proteins then react with BCA (bicinchoninic acid) (Pierce) toform a soluble purple complex presenting a high adsorbance at 562 nm,which is proportional to the amount of proteins present in the sample.

SDS-PAGE analysis of 3 purified bulks (FIG. 11) shows no difference inreducing and non reducing conditions (cf. lanes 2, 3 and 4 versus lanes5, 6 and 7). The pattern consists of a major band at 70 kDa, a smear ofhigher MW and faint degradation bands. All the bands are detected by aspecific anti P501S monoclonal antibody.

EXAMPLE V Vaccine Preparation Using CPC-P501S His Protein

The protein of Example 3 or 4 can be formulated into a vaccinecontaining QS21 and 3D-MPL in an oil in water emulsion.

1.—Vaccine Preparation:

The antigen produced as shown in Example 1 to 3 a C-LytA-P2-P501S His.As an adjuvant, the formulation comprises a mixture of 3 de-O-acylatedmonophosphoryl lipid A (3D-MPL) and QS21 in an oil/water emulsion. Theadjuvant system SBAS2 has been previously described WO 95/17210.

3D-MPL: is an immunostimulant derived from the lipopolysaccharide (LPS)of the Gram-negative bacterium Salmonella minnesota. MPL has beendeacylated and is lacking a phosphate group on the lipid A moiety. Thischemical treatment dramatically reduces toxicity while preserving theimmunostimulant properties (Ribi, 1986). Ribi Immunochemistry producesand supplies MPL to SB-Biologicals.

Experiments performed at Smith Kline Beecham Biologicals have shown that3D-MPL combined with various vehicles strongly enhances both the humoraland a TH1 type of cellular immunity.

QS21: is a natural saponin molecule extracted from the bark of the SouthAmerican tree Quillaja saponaria Molina. A purification techniquedeveloped to separate the individual saponins from the crude extracts ofthe bark, permitted the isolation of the particular saponin, QS21, whichis a triterpene glycoside demonstrating stronger adjuvant activity andlower toxicity as compared with the parent component. QS21 has beenshown to activate MHC class I restricted CTLs to several subunit Ags, aswell as to stimulate Ag specific lymphocytic proliferation (Kensil,1992). Aquila (formally Cambridge Biotech Corporation) produces andsupplies QS21 to SB-Biologicals.

Experiments performed at SmithKline Beecham Biologicals havedemonstrated a clear synergistic effect of combinations of MPL and QS21in the induction of both humoral and TH1 type cellular immune responses.

The oil/water emulsion is composed an organic phase made of of 2 oils (atocopherol and squalene), and an aqueous phase of PBS containing Tween80 as emulsifier. The emulsion comprised 5% squalene 5% tocopherol 0.4%Tween 80 and had an average particle size of 180 nm and is known as SB62(see WO 95/17210).

Experiments performed at SmithKline Beecham Biologicals have proven thatthe adjunction of this O/W emulsion to 3D-MPUQS21 (SBAS2) furtherincreases the immunostimulant properties of the latter against varioussubunit antigens.

2.—Preparation of Emulsion SB62 (2 Fold Concentrate):

Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2%solution in the PBS. To provide 100 ml two fold concentrate emulsion 5 gof DL alpha tocopherol and 5 ml of squalene are vortexed to mixthoroughly. 90 ml of PBS/Tween solution is added and mixed thoroughly.The resulting emulsion is then passed through a syringe and finallymicrofluidised by using an M110S microfluidics machine. The resultingoil droplets have a size of approximately 180 nm.

3.—Formulations:

A typical formulation containing 3D-MPL and QS21 in an oil/wateremulsion is performed as follows: 20 μg-25 μg C-LytA P2-P501S arediluted in 10 fold concentrated of PBS pH 6.8 and H₂O before consecutiveaddition of SB62 (50 μl), MPL (20 μg), QS21 (20 μg), optionallycomprising CpG oligonucleotide (100 μg) and 1 μg/ml thiomersal aspreservative. The amount of each component may vary as necessary. Allincubations are carried out at room temperature with agitation.

EXAMPLE VI Codon-Optimised P501S Sequences

1.—Generation of the Control Recombinant Plasmids:

Full-length P501S sequence was cloned into pVAC (Thomsen, Immunology,1998; 95:51OP105), generating expression plasmid JNW680. SEQ ID NO:17represents human P501S expression cassette in the plasmid JNW680 and isillustrated in FIG. 12. The protein sequence of SEQ ID NO:17 is shown insingle letter format, the start and stop codons being shown in bold. TheKozak sequence is denoted by the hash symbols. The codon usage index ofthe human P501S sequence (SEQ ID NO:17) is 0.618, as calculated by theSynGene programme.

SynGene Programme

Basically, the codons are assigned using a statistical method to givesynthetic gene having a codon frequency closer to that found naturallyin highly expressed E. coli and human genes.

SynGene is an updated version of the Visual Basic program calledCalcgene, written by R. S. Hale and G Thompson (Protein Expression andPurification Vol. 12 pp. 185-188 (1998). For each amino acid residue inthe original sequence, a codon was assigned based on the probability ofit appearing in highly expressed E. coli genes. Details of the Calcgeneprogram, which works under Microsoft Windows 3.1, can be obtained fromthe authors. Because the program applies a statistical method to assigncodons to the synthetic gene, not all resulting codons are the mostfrequently used in the target organism. Rather, the proportion offrequently and infrequently used codons of the target organism isreflected in the synthetic sequence by assigning codons in the correctproportions. However, as there is no hard-and-fast rule assigning aparticular codon to a particular position in the sequence, each time itis run the program will produce a different synthetic gene—although eachwill have the same codon usage pattern and each will encode the sameamino acid sequence. If the program is run several times for a givenamino acid sequence and a given target organism, several differentnucleotide sequences will be produced which may differ in the number,type and position of restriction sites, intron splice signals etc., someof which may be undesirable. The skilled artisan will be able to selectan appropriate sequence for use in expression of the polypeptide on thebasis of these features.

Furthermore, since the codons are randomly assigned on a statisticalbasis, it is possible (although perhaps unlikely) that two or morecodons which are relatively rarely used in the target organism might beclustered in close proximity. It is believed that such dusters may upsetthe machinery of translation and result in particularly low expressionrates, so the algorithm for choosing the codons in the optimized geneexcludes any codons with an RSCU value of less than 0.2 for highlyexpressed genes in order to prevent any rare codon clusters beingfortuitously selected. The distribution of the remaining codons is thenallocated according to the frequencies for highly expressed E. coli togive an overall distribution within the synthetic gene that is typicalsuch genes (coefficient=0.85) and also for highly expressed human genes(coefficient=0.50).

Syngene (Peter Ertl, unpublished), an updated version of the Calcgeneprogram, allows exclusion of rare codons to be optional, and is alsoused to allocate codons according to the codon frequency pattern ofhighly expressed human genes.

The sequence of the CPC-P501S cassette cloned from the vector pRIT15201(see FIG. 7) into pVAC, thereby generating plasmid JNW735, is set forthin SEQ ID NO:18 and is illustrated in FIG. 13. This sequence isidentical to the pRIT15201 sequence with the exception of the removal ofthe His tag and the addition of a Kozak sequence (GCCACC) andappropriate restriction enzyme sites. The amino acid sequence of SEQ IDNO:18 is shown in single letter format, the start and stop codons areshown in bold. The boxed residues are the P2 helper epitope of tetanustoxoid. The underlined residues are the Clyta purification tag. TheKozak sequence is denoted by the hash symbols.

2.—Generation of the Recombinant Plasmids with P501S Codon OptimisedSequences:

Although the codon coefficient index (CI) of P501S native sequence isalready high (0.618), it is possible increase the CI value further. Thiswill have two potential benefits—to improve the antigen expressionand/or immunogenicity and to reduce the possibility for recombinationbetween the P501S vector and genomic sequences.

Using the Syngene programme, a selection (SEQ ID NO:19 to SEQ ID NO:20)of codon optimised sequences was obtained (FIG. 14). Table 3 below showsa comparison of the codon coefficient index for the starting P501Ssequence and the two representative codon optimised sequences, selectedon the basis of a suitable restriction enzyme site profile and a good CIindex. TABLE 3 Comparison of the codon coefficient indices of two codonoptimised P501S genes Sequence Codon coefficient Index (CI) P501S 0.618SEQ ID NO: 19 0.725 SEQ ID NO: 20 0.7553. Further Evaluation of the Codon-Optimised SequencesSequence SEQ ID NO:19

Although SEQ ID NO: 19 has a good CI index (0.725), it contains adoublet of rare codons at amino acids position 202 and 203. These codonswere manually substituted with more frequent codons by changing the DNAsequence from TTGTTG to CTGCTG. To facilitate cloning and expression,restriction enzyme sites and a Kozak sequence were added. The finalengineered sequence (SEQ ID NO:21) is shown in FIG. 15. The Syngeneprogramme was used to fragment this sequence into oligonucleotides witha minimum overlap of 19-20 bases. Therefore, FIG. 15 shows there-engineered P501S codon optimised SEQ ID NO. 19. Restriction enzymesites are underlined, Kozak sequence is bolded, re-engineered DNAsequence to remove a rare codon doublet is boxed.

Using a two-step PCR protocol, the overlapping primers generated by theSyngene programme were first assembled using a PCR Assembly protocol(detailed below). The assembly reaction generates a diverse populationof fragments. The correct full-length fragment was recovered/amplifiedusing the PCR recovery protocol and the terminal primers. The resultingPCR fragment was excised from an agarose gel, purified, restricted withNheI and XhoI and cloned into pVAC. Positive clones were identified byrestriction enzyme analysis and confirmed by double-stranded sequencing.This generates plasmid JNW766, which, due to the error-prone nature ofthe PCR process, contained a single silent mutation (C to T at position360 of SEQ ID NO: 21).

1. Assembly Reaction—PCR Conditions, Generic Protocol

Reaction mix (total volume=50 μl):

-   -   1× Reaction buffer (Pfx or Proofstart)    -   1 μl Oligo pool (equal mix of all overlapping oligos)    -   0.5 mM dNTPs    -   DNA polymerase (Pfx or Proofstart, 2.5-5U)    -   +/−1 mM MgSO₄    -   +/−1× enhancer solution (Pfx enhancer or Proofstart buffer Q)        1. 94° C. for 120 s (Proofstart only)        2. 94° C. for 30 s        3. 40° C. for 120 s        4. 72° C. for 10 s        5. 94° C. for 15 s        6. 40° C. for 30 s        7. 72° C. for 20 s+3 s/cycle        8. Cycle to step 5, 25 times        9. Hold at 4° C.        2. Recovery Reaction—PCR Conditions (Generic Protocol)        Reaction mix (total volume=50 μl):    -   1× Reaction buffer (Pfx or Proofstart)    -   5-10 μl assembly reaction mix    -   0.3-0.75 mM dNTPs    -   50 pmol primer (5′ terminal primer, sense orientation)    -   50 pmol primer (3′ terminal primer, anti-sense orientation)    -   DNA polymerase (Pfx or Proofstart, 2.5-5U)    -   +/−1 mM MgSO₄    -   +/−1× enhancer solution (Pfx enhancer or Proofstart buffer Q)        1. 94° C. 120 s (Proofstart only)        2. 94° C. 45 s        3. 60° C. 30 s        4. 72° C. 120 s        5. Cycle to step 2, 25 times        6. 72° C. 240 s        7. Hold at 4° C.        Sequence SEQ ID NO:20

Although SEQ ID NO: 20 has a very good CI index (0.755), it was noticedthat it contained a doublet of rare codons at amino acids position 131and 132. These codons were manually substituted with more frequentcodons by changing the DNA sequence from TTGTTG to CTGCTG. To facilitatecloning, an internal BamHI site was removed by mutating G to C (see thedouble-underlined nucleotide in FIG. 16). To facilitate cloning andexpression, restriction enzyme sites and a Kozak sequence were added.The final engineered sequence (SEQ ID NO:22) is shown in FIG. 16. TheSyngene programme was used to fragment this sequence intooligonucleotides with a minimum overlap of 19-20 bases.

FIG. 16 therefore shows the re-engineered P501S codon optimised sequence20 (SEQ ID NO:22). Restriction enzyme sites are underlined, Kozaksequence is bolded, re-engineered DNA sequence to remove a rare codondoublet is boxed and a silent point mutation to remove a BamHI site isdouble-underlined.

Using a similar two-step PCR protocol to the one described above,full-length P501S fragment was amplified and cloned into pVAC. Positiveclones were identified by restriction enzyme analysis and confirmed bydouble-stranded sequencing. This generates plasmid JNW764. The sequenceof the P501S coding cassette is shown in FIG. 16 (SEQ ID NO: 22).

DNA Sequence Similarity

Pair distances following alignment by the ClustalV (weighted) method areshown in Table 3 below. Table 4 below shows percent similarity betweenthe starting human P501S sequence and the two codon optimised sequencesSEQ ID NO:21 and 22 selected for further investigation. The dataconfirms that the codon optimised DNA sequences are approximately 80%similar to the original P501S sequence. TABLE 4 SEQ ID NO: % similaritywith starting P501S sequence 21 79.6 22 79.4

EXAMPLE VII Codon-Optimised CPC Sequences

1.—Approach

Since the original CPC sequence was originally designed for optimalexpression in yeast, this section describes the process of codonoptimising for human expression.

2.—Sequence Design

The starting sequence for the optimisation of CPC is shown in FIG. 17(SEQ ID NO: 23). This is derived entirely from the pRIT15201 andcontains the entire coding sequence of CPC plus four amino acids ofP501S to facilitate downstream cloning. Using the Syngene programme, aselection of codon optimised sequences were obtained, from whichrepresentative sequences are shown in FIG. 18 (SEQ ID NO: 24-25). Table5 below shows a comparison of the codon coefficient index for thestarting CPC sequence and the two representative codon optimisedsequences. TABLE 5 Codon coefficient indices for two CPC optimisedsequences Sequence Codon coefficient index (CI) Original CPC = SEQ IDNO: 23 0.506 SEQ ID NO: 24 0.809 SEQ ID NO: 25 0.800

In addition to the codon optimisation, all sequences were also screenedfor restriction enzyme cloning sites. On the basis of the highest CIvalue and a favourable restriction enzyme site profile, SEQ ID NO: 24was selected for construction. To facilitate cloning and expression, 5′and 3′ cloning sites were added and a Kozak sequence (GCCACC) wasinserted 5′ of the initiating ATG start codon. This engineered sequenceis shown in FIG. 19 (SEQ ID NO:26). This sequence includes four aminoaicds of P501S (boxed), restriction enzyme cloning sites (NheI and XhoI,underlined), a Kozak sequence (Bold), a stop codon (italicised) and 4 bpof flanking irrelevant DNA to facilitate cloning.

The Syngene programme was used to fragment this sequence into 50-60-meroligonucleotides with a minimum overlap of 18-20 bases.

Using a similar two-step PCR protocol to the one described above, thecorrect fragment was recovered/amplified and cloned into pVAC. Positiveclones were identified by restriction enzyme analysis and sequenceverified generating vector JNW759.

4.—DNA Similarity

Pair Distances following alignment ClustalV (Weighted) are shown inTable 6 below. The table shows percent similarity at the DNA levelbetween the starting sequence of CPC and the codon optimised sequenceand confirms that the codon optimised sequences are approximately 80%similar to the original CPC sequence. TABLE 6 % similarity with SequenceSEQ ID NO: starting CPC sequence 24 80.2 25 81.6

EXAMPLE VII Construction of the P501S Fusion Candidate

All the candidates shown in the schematic below are codon optimised andconstructed using overlapping PCR methodologies from plasmids JNW764 andJNW759 as templates (SEQ ID NO: 22 and SEQ ID NO: 26 respectively), andcloned into the expression vector p7313 ie.

The four candidates shown schematically below are based upon CPC-P501S.Codon optimised CPC-P501S is construct A. Candidates B, C, D alsoinclude the sequence encoding the N terminal 50 amino acids of P501S,positioned either at the N terminus of CPC-P501S (construct D), the Cterminus of CPC-P501S (construct C), or between CPC and P501S (constructB). A schematic representation of the constructs is given in FIG. 20.

The nucleotide and protein sequence for each of the four constructs isshown in SEQ ID NO: 37-40 for the nucleotide sequences, and SEQ ID NO.45-48 for the corresponding polypeptide sequences. In constructs A, Cand D, the underlined codon preferentially encodes tyrosine (either TACor TAT) but the nucleotide sequence may be altered to encode threonine(either ACA, ACC, ACG or ACT). In construct B, the underlined codonpreferentially encodes threonine (either ACA, ACC, ACG or ACT), but thenucleotide sequence may be altered to encode tyrosine (either TAC orTAT). In all constructs, the coding sequence is flanked by appropriaterestriction enzyme cloning sites (in this case, NotI and BamHI), and aKozak sequence immediately upstream of the initiating ATG. Table 7 belowshows the plasmid identification for the constructs detailed above:TABLE 7 Amino acid at Construct underlined codon Sequence of codonPlasmid ID A Tyrosine TAC JNW771 B Threonine ACA JNW773 B Tyrosine TACJNW770 C Tyrosine TAC JNW777 D Tyrosine TAC JNW769

The cellular responses following immunisation with p7313-ie (emptyvector), pVAC-P501S (JNW735), JNW770, JNW771 and JNW773 were assessed byELISPOT following a primary immunisation by PMID at day 0 and threeboosts at day 21, 42 and 70. Assays were carried out 7 days post boost.FIG. 27 shows that good IL-2 ELISPOT responses were detected in miceimmunised with JNW770, JNW771 and JNW773.

EXAMPLE IX Immunogenicity Experiments Using Particle-MediatedIntra-Dermal Delivery (PMID) Studies

Full-length P501S, when delivered by particle mediated intra-dermaldelivery (PMID), generates good antibody & cellular responses. Thesedata demonstrate that the PMID is a very effective delivery route.Furthermore, comparison of P501S and CPC-P501S confirms that CPC-P501Sinduces a stronger immune response as determined by peptide ELISPOT.

1.—Materials & Methods

1.1. Cutaneous Gene Gun Immunisation

Plasmid DNA was precipitated onto 2 μm diameter gold beads using calciumchloride and spermidine. Loaded beads were coated onto Tefzel tubing asdescribed (Eisenbraum et al, 1993; Pertmer et al, 1996). Particlebombardment was performed using the Accell gene delivery system (PCT WO95/19799). For each plasmid, female C57BL/6 mice were immunised on days0, 21, 42 and 70. Each administration consisted of two bombardments withDNA/gold, providing a total dose of approximately 4-5 μg of plasmid.

1.2. ELISPOT Assays for T Cell Responses to the P501S Gene Product

a) Preparation of Splenocytes

Spleens were obtained from immunised animals at 7-14 days post boost.Spleens were processed by grinding between glass slides to produce acell suspension. Red blood cells were lysed by ammonium chloridetreatment and debris was removed to leave a fine suspension ofsplenocytes. Cells were resuspended at a concentration of 8×10⁶/ml inRPMI complete media for use in ELISPOT assays.

b) Screening of Peptide Library

A peptide library covering a majority of the P501S sequence was obtainedfrom Corixa Corp. The library contained fifty 15-20mer peptidesoverlapping by 4-11 amino acids peptides. The peptides are numbered1-50. In addition, a prediction programme (H-G. Rammensee, et al.:Immunogenetics, 1999, 50: 213-219)(http://syfpeithi.bmi-heidelberg.com/) was used to predict putative Kband Db epitopes from the P501S sequence. The ten best epitopes for Kband Db were ordered from Mimotopes (UK) and included in the library(peptides 51-70). For screening of the peptide library, peptides wereused at a final concentration of 50 μg/ml (approx. 25-50 μM) in IFNγ andIL-2 ELISPOTS using the protocol described below. For IFNγ ELISPOTS,IL-2 was added to the assays at 10 ng/ml. Splenocytes used for thescreening were taken at day 84 from C57BL/6 mice immunised at day 0, 21,42 and 70. Three peptides were identified from the libraryscreen—Peptides 18 (HCRQAYSVYAFMISLGGCLG), 22 (GLSAPSLSPHCCPCRARLAF) and48 (VCLAAGITYVPPLLLEVGV). These peptides were subsequently used in theELISPOT assays

c) ELISPOT Assay

Plates were coated with 15 μg/ml (in PBS) rat anti mouse IFNγ or ratanti mouse IL-2 (Pharmingen). Plates were coated overnight at +4° C.Before use the plates were washed three times with PBS. Splenocytes wereadded to the plates at 4×10⁵ cells/well. Peptides identified in thelibrary screen were re-ordered from Genemed Synthesis and used at afinal concentration of 50 μg/ml. CPC-P501S protein (GSKBio) was used inthe assay at 20 μg/ml. ELISPOT assays were carried out in the presenceof either IL-2 (10 ng/ml), IL-7 (10 ng/ml) or no cytokine. Total volumein each well was 200 μl. Plates containing peptide stimulated cells wereincubated for 16 hours in a humidified 37° C. incubator.

e) Development of ELISPOT Assay Plates.

Cells were removed from the plates by washing once with water (with 10minute soak to ensure lysis of cells) and three times with PBS. Biotinconjugated rat anti mouse IFNg or IL-2 (Phamingen) was added at 1 μg/mlin PBS. Plates were incubated with shaking for 2 hours at roomtemperature. Plates were then washed three times with PBS beforeaddition of Streptavidin alkaline phosphatase (Caltag) at 1/1000dilution. Following three washes in PBS spots were revealed byincubation with BCICP substrate (Biorad) for 15-45 mins. Substrate waswashed off using water and plates were allowed to dry. Spots wereenumerated using an image analysis system devised by Brian Hayes, AsthmaCell Biology unit, GSK.

1.3. ELISA Assay for Antibodies to the P501S Gene Product

Serum samples were obtained from the animals by venepuncture on days −1,28, 49 and 56, and assayed for the presence of anti-P501S antibodies.ELISA was performed using Nunc Maxisorp plates coated overnight at 4° C.with 0.5 μg/ml of CPC-P501S protein (GSKBio) in sodium bicarbonatebuffer. After washing with TBS-Tween (Tris-buffered saline, pH 7.4containing 0.05% of Tween 20) the plates were blocked with Blockingbuffer (3% BSA in TBS-Tween buffer) for 2 hrs at room temperature. Allsera were incubated at 1:100 dilution for 1 hr at RT in Blocking buffer.Antibody binding was detected using HRP-conjugated rabbit anti-mouseimmunoglobulins (#P0260, Dako) at 1:2000 dilution in Blocking buffer.Plates were washed again and bound conjugate detected using Fast OPDcolour reagents (Sigma, UK). The reaction was stopped by the addition of3M sulphuric acid, and the OPD product quantitated by measuring theabsorbance at 490 nm.

1.4. Transient Transfection Assays

Human P501S expression from various DNA constructs was analysed bytransient transfection of the plasmids into CHO (Chinese hamster ovary)cells followed by Western blotting on total cell protein. Transienttransfections were performed with the Transfectam reagent (Promega)according to the manufacturer's guidelines. In brief, 24-well tissueculture plates were seeded with 5×10⁴ CHO cells per well in 1 ml DMEMcomplete medium (DMEM, 10% FCS, 2 mM L-glutamine, penicillin 100 IU/ml,streptomycin 10 μg/ml) and incubated for 16 hours at 37° C. 0.5 μg DNAwas added to 25 μl of 0.3M NaCl (sufficient for one well) and 2 μl ofTransfectam was added to 25 μl of Milli-Q. The DNA and Transfectamsolutions were mixed gently and incubated at room temperature for 15minutes. During this incubation step, the cells were washed once in PBSand covered with 150 μl of serum free medium (DMEM, 2 mM L-glutamine).The DNA-Transfectam solution was added drop wise to the cells, the plategentle shaken and incubated at 37° C. for 4-6 hours. 500 μl of DMEMcomplete medium was added and the cells incubated for a further 48-72hours at 37° C.

2. Western Blot Analysis of CHO Cells Transiently Transfected with P501SPlasmids

The transiently transfected CHO cells were washed with PBS and treatedwith a Versene (1:5000)/0.025% trypsin solution to transfer the cellsinto suspension. Following trypsinisation, the CHO cells were pelletedand resuspended in 50 μl of PBS. An equal volume of 2×NP40 lysis bufferwas added and the cells incubated on ice for 30 minutes. 100 μl of 2×TRIS-Glycine SDS sample buffer (Invitrogen) containing 50 mM DTT wasadded and the solution heated to 95° C. for 5 minutes. 1-20 μl of samplewas loaded onto a 4-20% TRIS-Glycine Gel 1.5 mm (Invitrogen) andelectrophoresed at constant voltage (125V) for 90 minutes in 1×TRIS-Glycine buffer (Invitrogen). A pre-stained broad range marker (NewEngland Biolabs, #P7708S) was used to size the samples. Followingelectrophoresis, the samples were transferred to Immobilon-P PVDFmembrane (Millipore), pre-wetted in methanol, using an Xcell III BlotModule (Invitrogen), 1× Transfer buffer (Invitrogen) containing 20%methanol and a constant voltage of 25V for 90 minutes. The membrane wasblocked overnight at 4° C. in TBS-Tween (Tris-buffered saline, pH 7.4containing 0.05% of Tween 20) containing 3% dried skimmed milk (Marvel).The primary antibody (10E3) was diluted 1:1000 and incubated with themembrane for 1 hour at room temperature. Following extensive washing inTBS-Tween, the secondary antibody (HRP-conjugated rabbit anti-mouseimmunoglobulins (#P0260, Dako)) was diluted 1:2000 in TBS-Tweencontaining 3% dried skimmed milk and incubated with the membrane for onehour at room temperature. Following extensive washing, the membrane wasincubated with Supersignal West Pico Chemiluminescent substrate (Pierce)for 5 minutes. Excess liquid was removed and the membrane sealed betweentwo sheets of cling film, and exposed to Hyperfilm ECL film(Amersham-PharmaciaBiotech) for 1-30 minutes.

3. Generation of the Full-Length Human P501S Expression Cassette

The starting point for the construction of a P501S expression cassettewas the plasmid pcDNA3.1-P501S (Corixa Corp), which has a pcDNA3.1backbone (Invitrogen) containing a full-length human P501S cDNA cassettecloned between the EcoRI and NotI sites. This vector is also termedJNW673. The presence of P501S was confirmed by fluorescent sequencing.The sequence of the cDNA cassette is given by the NCBI/Genbank sequence(accession number AY033593). Human P501S was PCR amplified from JNW673template DNA, restricted with XbaI and SalI and cloned into theNheI/XhoI sites of pVAC generating vector JNW680. The correctorientation of the fragment relative to the CMV promoter was confirmedby PCR and by DNA sequencing. The sequence of the expression cassette isshown in FIG. 12 (SEQ ID NO: 17).

To construct a CPC-P501S expression cassette, CPC-P501S was PCRamplified from the vector pRIT15201 (see FIG. 7), restricted with XbaIand SalI and cloned into the NheI and XhoI sites of pVAC, generatingplasmid JNW735. The correct orientation was confirmed by PCR andsequencing. The sequence of the CPC-P501S expression cassette is shownin FIG. 13 (SEQ ID NO:18).

4. Expression of Human P501S from Plasmids JNW680 and JNW735

The P501S expression plasmids were transiently transfected into CHOcells and a total cell lysate prepared as described in methods. AWestern blot of a total cell lysate identified single bands ofapproximately 55 kDa and 62 kDa for samples transfected with JNW680 andJNW735 respectively (FIG. 21). This is consistent with the predictedmolecular weights of 59.3 kDa and 63.3 kDa for P501S and CPC-P501Srespectively. The addition of the CPC tag does not adversely affect theexpression of P501S.

5. Results

5.1. Antibody Responses to Human P501S Following PMID Immunisation

The antibody responses following immunisation with pVAC (empty vector)and pVAC-P501S (JNW680) were assessed by ELISA following a primaryimmunisation by PMID at day 0 and three boosts at day 21 and day 42 andday 70. FIG. 22 shows the antibody responses from sera taken at day −1,day 28 and day 49 (mice A1-3, B1-3) and day 56 (mice A4-6, B4-9). Whilstthere were some non-specific responses to the pVAC empty vector,specific responses to the P501S construct were seen in 5 of 9 mice.

5.2. Identification of novel T cell epitopes from human P501S in C57BL/6mice by screening of a P501S peptide library Following immunisation withJNW680 (pVAC-P501S) by PMID at day 0 and three boosts at day 21 and day42 and day 70, ELISPOT assays were carried out at day 84. Peptides fromthe P501S library were tested at 50 μg/ml final concentration. From thisinitial screen, three peptides were found to stimulate IFNγ and/or IL-2secretion. Peptides 18, 22 and 48 (FIG. 23). These peptides were used insubsequent cellular assays.

5.3. Cellular Responses to pVAC-P501S (JNW680) Following PMIDImmunisation

The cellular responses following immunisation with pVAC (empty vector)and pVAC-P501S were assessed by ELISPOT following a primary immunisationby PMID at day 0 and three boosts at day 21, 42 and 70. Assays werecarried out 7 days post boost. Two different assay conditions wereused: 1) Peptides 18, 22 and 48 identified in the peptide library screenused at 50 μg/ml final concentration and 2) CPC-P501S protein used at 20μg/ml final concentration. FIG. 24A shows that whilst there were noP501S-specific responses to the empty vector (A4-6), the pVAC-P501Sconstruct induced specific IFN-γ responses to Peptides 18 and 22 in allmice (B6-9) whilst one mouse (B7) also showed an IFN-γ response toPeptide 48. FIG. 24B shows that all mice showed specific IL-2 responsesto Peptides 18, 22 and 48. Furthermore, pVAC-P501S immunised mice (B6-9)also showed moderate IL-2 responses to CPC-P501S, whereas the emptyvector immunised mice (A4-6) showed no responses.

5.4. Comparison of Cellular Responses to P501S and CPC-P501S FollowingPMID Immunisation.

The cellular responses following immunisation with pVAC (empty vector),pVAC-P501S (JNW680) and CPC-P501S (JNW735) were assessed by ELISPOTfollowing a primary immunisation by PMID at day 0 and boosts at day 21and 42. Assays were carried out 7 days post boost. Two different assayconditions were used: 1) Peptides 18, 22 and 48 identified in thepeptide library screen used at 50 μg/ml final concentration and 2)CPC-P501S protein used at 20 μg/ml final concentration. FIG. 25 showsthat at day 28, CPC-P501S induced good IL-2 responses to 10 μg/ml ofpeptide 22, whilst there were no P501S-specific responses to either theempty vector or the pVAC-P501S. These results were also seen usingCPC-P501S protein to re-stimulated the splenocytes. At day 49 (post2^(nd) boost), the responses induced by P501S and CPC-P501S wereequivalent. These data suggest that the addition of the CPC tag improvesthe kinetics and/or magnitude of the response to P501S.

EXAMPLE IX Immunogenicity Experiments in Mice Using P501SProtein+Adjuvant Studies

1. Design and Adjuvant Formulation

The immune response induced by vaccination using the recombinantpurified CPC-P501S protein formulated in adjuvants is characterized inexperiments performed in mice. Groups of 5 to 10, eight weeks old femaleC57BL6 mice are vaccinated, 2-6 times intra-muscularly at 2 weeksintervals with 10 μg of the CPC-P501S protein formulated in differentadjuvant systems. The volume administered corresponds to 1/10^(th) of ahuman dose (50 μl).

The serology (total Ig response) and cellular response (T celllymphoproliferation and cytokine production) are analyzed on spleencells, 6-14 days after the last vaccination using standard protocols asdescribed in Gerard, c. et al, 2001, Vaccine 19, 2583-2589.

The data of one representative experiment is shown. It included 5 groupsof eight C57BL/6 female mice which received 4 intramuscular injectionsof CPC P501 (10 μg)+adjuvant (A, B, C) at days 0, 14, 28, 42. Example Vprovides an experimental protocol of how to carry out the formulations.Briefly the adjuvant formulations are as follows (quantities given forone dose of 100 μl)):

-   -   Adjuvant A: QS21 (10 μg), MPL (10 μg) and CpG7909 (100 μg) made        according to the method disclosed in WO 00/62800;    -   Adjuvant B: formulation of QS21 (20 μg), MPL (20 μg), CpG7909        (100 μg) and 50 μf SB62 oil-in-water emulsion (WO 95/17210);    -   Adjuvant C: formulation of QS21 (10 μg), MPL (10 μg), CpG7909        (100 μg) and 10 μl SB62 oil-in-water emulsion (WO 99/12565).        2. Serology

The total Ig response induced by vaccination was measured by ELISA usingeither the CPC-P501 or RA12-P501 (C term, which is a truncated form ofthe P501 protein corresponding to the C terminus of the protein fused atits N terminus, to a TB derived protein RA12-Ra12 is derived from MTB32Aantigen described in Skeiky et al., Infection and Immun. (1999)67:3998-4007).

The adjuvanted CPC-P501S proteins give a good antibody response aftervaccination.

3. Cellular Response

3.1. Lymphoproliferation

7 days after the latest vaccine, lymphoproliferation was performed onspleen cells individually. 2.10e5 spleen cells were plated inquadruplicate, in 96 well microplate, in RPMI medium containing 1%normal mice serum. After 72 hours of restimulation with either theimmunogen (CPC-P501) or the truncated protein (RA12 P501) at differentconcentration, 1 μCi 3H thymidine (Amersham 5 Ci/ml) was added. After 16hours, cells were harvested onto filter plates. Incorporatedradioactivity was counted in a β counter. Results are expressed in CPMor as stimulation indexes* (geomean CPM in cultures with antigen/geomeanCPM in cultures without antigen).

Re-stimulation with ConA (2 μg/ml) as positive control was included aspositive control.

As shown in FIG. 26, a P501 specific lymphoproliferation is seen in thespleen of all groups of mice receiving the adjuvanted protein after invitro re-stimulation with either the immunogen or another P501 proteinmade in another expression system (E. coli), indicating that T cellshave been primed in vivo by the vaccination.

3.2. IFNg Production Measured by Intracellular Staining of Spleen Cells

Bone Marrow Dendritic Cells (BMDC) obtained after culture of mouse PBLfor 7 days in the presence of GMCSF.

7 days after the latest vaccine, spleen or PBL are collected and a cellsuspension prepared. 10e6 cells (1 pool per group) were incubated +/−18hrs with 10e5 BMDC pulsed overnight with 10 μg/ml of either the CPCp501protein or the RA12.

After a treatment with the 2.4.G.2 antibody, spleen cells were stainedwith fluorescent anti CD4 and CD8 antibodies (anti CD4-APC and an antiCD8PerCP). After a permeabilization and fixation step, cells werestained with a fluorescent anti IFNg-FITC antibody.

In mice vaccinated with CPC P501 in different adjuvant, both CD4 and CD8T cells are shown to produce IFNg in response to DC pulsed with eitherthe immunogen and the C-term p501 made in E. coli (as shown byintracellular straining of spleen and PBLs). There is an increase of4-10× in the % of cells making this cytokine in the groups receiving theadjuvanted CPC-P501S compared to the protein alone, and between 0.1 to10% of CD4 or CD8 T cells are shown to produce IFNg.

In conclusion, these data allow to conclude that the adjuvanted CPC-P501protein is immunogenic in mice.

Both a P501 specific humoral and cellular responses including IFNgproduction by CD4 and CD8 T cells can be detected after severalintramuscular vaccination with CPC P501 in adjuvants.

EXAMPLE X CPC-MUC-1 Constructs and Sequences

CPC sequence is taken from nucleotide SEQ ID NO. 28.

MUC1 sequence is available from Genbank database (accession numberNM_(—)002456).

1. MUC1-CPC Construct

Due to the presence of a signal sequence in MUC1 that is cleavedpost-translationally, the CPC motif was placed at the C-terminus. Theresulting MUC1-CPC DNA sequence is depicted in SEQ ID NO. xx (FIG. 28A)and the corresponding MUC1-CPC protein sequence in SEQ ID NO. yy (FIG.28B).

2. ss-CPC-MUC1 Construct

Due to the presence of a signal sequence in MUC1 that is cleavedpost-translationally, the MUC1 signal sequence was replaced by aheterologous leader sequence (from the human immunoglobulin heavy chain)and the CPC motif was inserted between the heterologous leader sequenceand the MUC1 sequence, generating a sequence termed ss-CPC-MUC1 asdepicted in FIG. 29.

1. A fusion partner protein comprising a choline binding domain and aheterologous promiscuous T helper epitope.
 2. A fusion partner proteinaccording to claim 1 wherein the choline binding domain is derived fromthe C terminus of LytA.
 3. A fusion partner protein according to claim 2wherein the C-LytA or derivatives comprises at least four repeats of anyof SEQ ID NO: 1 to
 6. 4. A fusion partner protein according to claim 1,wherein the choline binding domain is selected from the group of: a) theC-terminal domain of LytA as set forth in SEQ ID NO:7; b) the sequenceof SEQ ID NO:8; c) a peptide sequence comprising an amino acid sequencehaving at least 85% identity to any of SEQ ID NO:1 to 6; and d) apeptide sequence comprising an amino acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous amino acids from the amino acidsequence of SEQ ID NO:7 or SEQ ID NO:8.
 5. A fusion partner protein asclaimed in claim 1 further comprising a heterologous protein.
 6. Afusion protein as claimed in claim 5 wherein the heterologous protein ischemically conjugated the fusion partner.
 7. A fusion protein as claimedin claim 5 wherein the heterologous protein is derived from an organismselected from the following group: Human Immunodeficiency virus HIV-1,human herpes simplex viruses, cytomegalovirus, Rotavirus, Epstein Barrvirus, Varicella Zoster Virus, hepatitis A virus, hepatitis C virus,hepatitis E virus, from Respiratory Syncytial virus, parainfluenzavirus, measles virus, mumps virus, human papilloma viruses,flaviviruses, and Influenza virus, from Neisseria spp, Moraxella spp,Bordetella spp; Mycobacterium spp., M. tuberculosis; Escherichia spp,enterotoxic E. coli; Salmonella spp,; Listeria spp; Helicobacter spp;Staphylococcus spp., S. aureus, S. epidermidis; Borrelia spp; Chlamydiaspp., C. trachomatis, C. pneumoniae; Plasmodium spp., P. falciparum;Toxoplasma spp., or Candida spp.
 8. A fusion protein as claimed in claim5 wherein the heterologous protein is a tumour associated protein ortissue specific protein or immunogenic fragment thereof.
 9. A fusionprotein as claimed in claim 8 wherein the heterologous protein orfragment thereof is selected from MAGE 1, MAGE 3, MAGE 4, PRAME, BAGE,LAGE 1, LAGE 2, SAGE, HAGE, XAGE, PSA, PAP, PSCA, prostein, P501S,HASH2, Cripto, B726, NY-BR1.1, P510, MUC-1, Prostase, STEAP, tyrosinase,telomerase, survivin, CASB616, P53, or her 2 neu.
 10. A fusion proteinas claimed in claim 6 further comprising an affinity tag of at least 4histidine residues.
 11. A nucleic acid sequence encoding a protein ofclaim
 1. 12. An expression vector comprising a nucleic acid sequence ofclaim
 11. 13. A host cell transformed with an expression vector of claim12.
 14. An immunogenic composition comprising a protein as claimed inany of claim 1 and a pharmaceutically acceptable excipient.
 15. Animmunogenic composition as claimed in claim 14 which additionallycomprises a TH-1 inducing adjuvant.
 16. An immunogenic composition asclaimed in claim 15 in which the TH-1 inducing adjuvant is selected fromthe group of adjuvants comprising: 3D-MPL, QS21, a mixture of QS21 andcholesterol, a CpG oligonucleotide or a mixture of two or more saidadjuvants.
 17. A process for the preparation of a immunogeniccomposition, comprising admixing the fusion protein of claim 6 with asuitable adjuvant, diluent or other pharmaceutically acceptable carrier.18. A process for producing a fusion protein of claim 1 comprisingculturing a host cell comprising a vector encoding said fusion proteinunder conditions sufficient for the production of said fusion proteinand recovering the fusion protein from the culture medium.
 19. Apharmaceutical composition comprising a fusion protein of claim
 1. 20.(canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)25. (canceled)
 26. A method of treating a patient suffering from cancerby administrating a safe and effective amount of a composition accordingto claim
 12. 27. A method according to claim 26 wherein said cancer isprostate cancer, colorectal cancer, lung cancer, breast cancer ormelanoma.
 28. An immunogenic composition comprising a DNA sequence asclaimed in claim 11 and a pharmaceutically acceptable excipient.
 29. Aprocess for the preparation of an immunogenic composition, comprisingadmixing the fusion protein of a polynucleotide of claim 11 with asuitable adjuvant, diluent or other pharmaceutically acceptable carrier.30. A method of eliciting an immune response in a patient comprisingadministering an immunogenic composition of claim
 14. 31. The methodaccording to claim 30, wherein said immune response is to be elicited bysequential administration of i) the said protein followed by a nucleicacid encoding said protein; or ii) a nucleic acid encoding said proteinfollowed by said protein.
 32. The method according to claim 31 whereinsaid nucleic acid sequence is coated onto biodegradable beads ordelivered via a particle bombardment approach.
 33. The method accordingto claim 31 wherein said protein is adjuvanted.
 34. The method accordingto claim 31 wherein the patient is suffering from or susceptible tocancer.
 35. The method according to claim 34 wherein said cancer isprostate cancer, colon cancer, lung cancer, breast cancer or melanoma.